Polyionene transformed modified polysaccharide supports

ABSTRACT

Polyionene-transformed modified polymer-polysaccharide separation matrix and use thereof in removing contaminants of microorganism origin from biological liquids are disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation-in-part of application Ser.No. 576,448, filed Feb. 2, 1984 now U.S. Pat. No. 4,663,163, which inturn is a continuation-in-part of application Ser. No. 466,114, filedFeb. 14, 1983 now abandoned. Further, the application is related toapplication Ser. No. 723,691, filed Apr. 16, 1985 now U.S. Pat. No.4,675,104, (which is a continuation-in-part of Ser. No. 633,904, filedJan. 23, 1985, abandoned, which is a continuation of Ser. No. 505,532,filed June 17, 1983, now U.S. Pat. No. 4,496,461) and application Ser.No. 758,036, filed concurrently herewith. These patent applications areincorporated by reference herein.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to separation matrices useful for removingmicroorganism-originated contaminants from biological liquids andmethods for their preparation and use. The separation matrices may beused as chromatographic separation media as well as providing the addedbenefit of antimicrobial activity and are characterized by the presenceof immobilized polyionene.

2. Brief Description of the Background Art

The broad applicability of ion exchange chromatography, which rangesfrom separation of inorganic and organic ions to that of proteinmolecules and other biomolecules, has made it a powerful and versatiletool for chemical and biochemical separations. The technique wasoriginally limited to the use of natural products such as cellulose,clay and other minerals containing mobile ions that would exchange withionic materials in the surrounding solute phase. Because of the lowexchange capacity of these natural products, however, practicalutilization thereof was limited, and synthetic organic polymers capableof exchanging ions were developed.

Among the first generation of synthetic ion exchange materials were theion exchange resins. The fundamental framework of these ion exchangeresins is an elastic three-dimensional hydrocarbon network comprisingionizable groups, either cationic or anionic, chemically bonded to thebackbone of a hydrocarbon framework. The network is normally fixed,insoluble in common solvents and is chemically inert. The ionizablefunctional groups attached to the matrix carry active ions which canreact with or can be replaced by ions in the solute phase. Therefore,the ions in the solute phase can be easily exchanged for the ionsinitially bound to the polymeric resins. Typical examples ofcommercially available ion exchange resins are the polystyrenescross-linked with DVB (divinylbenzene), and the methacrylatescopolymerized with DVB. In the case of polystyrene, a three-dimensionalnetwork is formed first, and the functional groups are then introducedinto benzene rings through chloromethylation. Since ion exchange resinsare elastic three-dimensional polymers, they have no definite pore size;only a steadily increasing resistance to flow of the polymer networklimits the uptake of ions and molecules of increasing size.

The resistance to flow exhibited by these resins is controlled by thedegree of crosslinking. With a low degree of crosslinking, thehydrocarbon network is more easily stretched, the swelling is large, andthe resin exchanges small ions rapidly and even permits relatively largeions to undergo reaction Conversely, as the crosslinking is increased,the hydrocarbon matrix is less resilient, the pores of the resin networkare narrowed, the exchange process is slower, and the exchangerincreases its tendency to exclude large ions from entering thestructure. The ion exchange resins made from polymeric resins have beensuccessfully applied for the removal of both organic and inorganic ionsfrom aqueous media but they are normally unsuitable for the separationof biopolymers such as proteins. This is due, among others, to thefollowing reasons:

(1) The highly crosslinked structure of the resins has rather narrowpores to accommodate the diffusion of proteins; the proteins thereforeare virtually restricted to the macrosurface area of the beads withconsequent limitation of solute loadings;

(2) The high charge density close to the proximity of the resin surfaceis unsuitable, since it causes excessive binding and distortion ofprotein structure;

(3) The hydrocarbon matrix is usually hydrophobic and is potentiallydangerous to the subtle three-dimensional structure of biopolymers,often causing denaturation of proteins.

The next generation of chromatographic materials useful for separationof proteins and other labile biological substances was based oncellulose ion exchangers. These lacked nonspecific adsorption and hadpracticable pore structure. Such prior art ion exchange celluloses aremade by attaching substituent groups with either basic or acidicproperties to the cellulose molecule by esterification, etherification,or oxidation reactions. Examples of cationic exchange celluloses arecarboxymethylated cellulose (CM), succinic half esters of cellulose,sulfoethylated cellulose, and phosphorylated cellulose. Examples ofanionic exchange celluloses are diethylaminoethyl cellulose (DEAE), andtriethylaminoethyl cellulose (TEAE). Ion exchange materials based oncellulose as the principal backbone or anchoring polymer, however, havenot enjoyed complete success due primarily to an inherent property ofcellulose: its affinity for water. Thus, prior art ion exchangematerials based on cellulose, while typically having high exchangecapacity, are difficult to use as a consequence of their tendency toswell, gelatinize or disperse on contact with an aqueous solution. Anideal ion exchange material should minimally interact with the solventsystem which carries the ions in solution through its pores; only inthis manner is it possible to obtain a rapid, free flowing ion exchangesystem.

A third generation of ion exchange materials, which were developed tosolve some of these problems, were the ion exchange gels. There gelscomprise large pore gel structures and include the commercially knownmaterial Sephadex®, which is a modified dextran. The dextran chains arecrosslinked to give a three-dimensional polymeric network. Thefunctional groups are attached by ether linkages to the glucose units ofthe dextran chains. Proteins are not denatured by the hydrophilicpolymeric network. Sephadex® exhibits very low nonspecific adsorption,which makes it ideal as a matrix for biological separations. However,the porosity of ion exchange gels is critically dependent on itsswelling properties, which in turn is affected by the environmentalionic strength, pH and the nature of the counter-ions. Swelling of gelsin buffer is caused primarily by the tendency of the functional groupsto become hydrated. The amount of swelling is directly proportional tothe number of hydrophilic functional groups attached to the gel matrix,and is inversely proportional to the degree of crosslinking present inthe gel. This characteristic swelling is a reversible process, and atequilibrium there is a balance between two forces: (1) the tendency ofthe gel to undergo further hydration, and hence to increase the osmoticpressure within the gel beads, and (2) the elastic forces of the gelmatrix. The osmotic pressure is attributed almost entirely to thehydration of the functional groups, and, since different ions havedifferent degrees of hydration, the particular counter-ions in an ionexchange gel can be expected to have a considerable influence upon thedegree of swelling. Since the pH, the electrolyte concentration and thenature of the counter-ions can all affect the hydration, leading to adifferent degree of gel swelling, the pore size in the gels is not inwell defined form but is rather dependent on the environmentalconditions. Gels without crosslinking provide large pores and highcapacity due to maximum swelling. They suffer, however, from theweakness of structural integrity and can easily be crushed with aminimum amount of pressure. Removal of the solvent from the gels oftenresults in collapse of the matrix. Highly crosslinked gels havemechanical strength, but lose capacity and pore size due to restrictionsin swelling.

Ion exchange gels made from synthetic polymers have also been used, andthey include crosslinked polyacrylamide (Bio-Gel P®), microreticularforms of polystyrene (Styragel®), poly(vinyl acetate) (Merck-o-Gel OR®),crosslinked poly(2-hydroxy ethylmethacrylate)(Spheron®), andpolyacryloylmorpholine (Enzacryl®). All of these follow the generaltrend: it may be possible to obtain dimensional stability with high flowrate or, alternatively, high capacity with swelling. It is, however, notpossible to obtain both capacity and high flow rate at the same time.

The failure of single components to have both capacity and dimensionalstability led to yet another generation of ion exchange materialscomprising composite structures, e.g., hybrid gels. Hybrid gels are madeby combining a semi-rigid component, for the purpose of conferringmechanical stability, with a second component, a softer network, whichis responsible for carrying functional groups. Agarose gel, which wouldotherwise be very soft and compressible, can be made stronger throughhybridizing with crosslinked polyacrylamide. The crosslinkedpolyacrylamide component is mechanically stronger than the agarose,improves the gel flow properties, and reduces the gel swelling, but itsacrifices molecular fractionation range. Examples of hybrid gels otherthan polyacrylamide/agarose (Ultrogels®), are polyacryloylmorpholine andagarose (Enzacryl®), and composite polystyrenes with large porepolystyrenes as a framework filled with a second type of lightlycrosslinked polymer.

Yet another composite gel structure is achieved by combining inorganicmaterials coated with organics, and are the types known as Spherosil®.Porous silica beads are impregnated with DEAE dextran so that theproduct will have the mechanical properties of silica, with the ionexchange properties of DEAE dextrans. These composites, however, havesevere channeling defects arising out of particle packing, and they havecapacity limitations on the coated surfaces.

Totally rigid inorganic supports such as porous silica or porous glasswhich are not susceptible to degradation have also been used to providehigh porosity, and high flow rate systems. The major problem, however,is nonspecific adsorption of proteins due to the silanol groups on thesilica surface. Since the hydrolysis of silica is directly related tothe pH conditions, the nonspecific adsorption by silica is minimal atneutral pH, but increases as the pH changes both to the acidic oralkaline ranges. A monolayer coating by either hydrophilic organicpolymers or silanization has been used in an attempt to overcome thisproblem.

In the technique of affinity chromatography, which enables the efficientisolation of biological macromolecules or biopolymers, by utilizingtheir recognition sites for certain supported chemical structures with ahigh degree of selectivity, the prior art has also utilized materials ofvarying chemical structure as supports. For example, agarose gels andcrosslinked agarose gels have been the most widely used supportmaterials. Their hydrophilicity makes them relatively free ofnonspecific binding, but their compressibility makes them lessattractive as carriers in large scale processing, such as inmanufacturing. Controlled-pore glass (CPG) beads have also been used inaffinity chromatography. Although high throughputs can be obtained withcolumns packed with CPG, this carrier is even more expensive thanagarose gel beads. Cellulose particles have also been used byimmunochemists for synthetic affinity sorbents. However, compared toagarose gels, cellulose particles are formed with more difficulty andtherefore, have received less attention in the preparation of affinitysorbents for enzymes. Cellulose, however, is perhaps the least expensiveof all support matrices. Two lesser used support matrices arepolyacrylamide gel beads and Sephadex® gel beads made from dextran andepichlorohydrin. Although convenient methods have been developed forusing them, the softness of these beads yields poor column packings, andtheir low molecular porosity yields a sorbent with poor ligandavailability to the ligate.

Coupek et al., U.S. Pat. No. 4,281,233 show supports for affinitychromatography which comprise copolymers of hydroxy alkyl acrylates ormethacrylates with crosslinking monomers. The copolymers containcovalently attached mono- or oligosaccharides. (An oligosaccharide isdefined in the art as having up to nine saccharide units. See, e.g.,Roberts, J. D., and Caserio, M. C., Basic Principles of OrganicChemistry, 1964, p. 615.)

A carrier for bio-active materials is also disclosed in Nakashima etal., U.S. Pat. No. 4,352,884. The Nakashima carrier comprises asubstrate coated with a copolymer. The substrate may be one of variousmaterials, including inorganic materials such as glass, silica, alumina,synthetic high polymers such as polystyrene, polyethylene and the like,and naturally occurring high polymers such as cellulose. The copolymeris made of a hydrophilic acrylate or methacrylate monomer which is ahydroxy or alkoxy alkyl acrylate or methacrylate, and a copolymerizableunsaturated carboxylic acid or amine. The base material or substrate iscoated with the copolymer by conventional coating or depositionprocedures, such as spraying, dipping, phase separation or the like. Thecopolymer may also contain small amounts of a crosslinking agent such asglycidyl acrylate or methacrylate. The crosslinking agent allows forcross-linking treatment after the coating process, and provides for theprevention of elution (presumably of the bioactive materials) from thecoating layer. The amounts of cross-linking agent are quite small, andrange between 0.5 and 1 percent by weight of the total copolymer weight.Such amounts of cross-linking agent are insufficient to causesubstantial covalent bonding or grafting of the copolymer onto theunderlying substrate. The copolymer in Nakashima is thus essentiallyonly physically coating the underlying substrate. Physical coating,however, is accompanied by a series of problems. The carrier would notbe expected to have an even distribution of the copolymer, would show amultilayered structure, and may have a possible uneven distribution offunctional groups.

Another reference of interest is Kraemer, U.S. Pat. No. 4,070,348, whichshows copolymers of glycidyl- and amino-containing acrylates, useful ascarriers for biologically active substances, such as polysaccharides,enzymes, peptides, hormones, etc. The structure of the final product inKraemer is that of an acrylic copolymer chain covalently modified at amultiplicity of sites thereon with substances such as enzymes, proteins,and the like.

This review of the prior art, its advantages and drawbacks, leads to theconclusion that there exists a need for a support useful both for ionexchange and affinity chromatography-based purification which will havehigh stability, high porosity, low nonspecific adsorption, high flowrate, poor compressibility, controlled gelation, and which will beuseful for industrial-scale biological separations. It is at theindustrial level of manufacturing, especially, where the aforementioneddrawbacks have had their most important effect and where this need isthe strongest.

Industrial scale molecular separation materials comprising fibrousmatrices with particulate immobilized therein have been described incommonly assigned U.S. Pat. No. 4,384,957 to Crowder, which is hereinincorporated by reference. This patent describes a composite fibermaterial formed by wet layering a sheet from an aqueous slurry ofparticulate, small refined fiber pulp and long soft fiber pulp. Thepurpose of the soft long fiber is physically to hold clumps ofparticulate material and refined pulp together. Sheets are formed from awet slurry by vacuum filtration, wherein the long fibers form in a planewhich is perpendicular to the direction of flow of the chromatographiccarrier fluid. This permits channels to form in the composite materialwhich are perpendicular to the direction of flow of the chromatographiccarrier fluid and allows these materials to serve as internal flowdistributors. The resulting matrix structure has proven to be aneffective way of eliminating channeling defects through internal flowdistribution mechanisms.

It is inevitable in prior art wet slurrying processes with slurriescomprising cationic materials, to obtain materials having unevendistribution of charges, wherein multilayer coating may occur in onespot, whereas other spots on the surface may be bare. Such products areacceptable in filtration processes due to the fact that the amount ofimpurities needed to be removed is relatively small compared to the bulkliquid volume, and that uneven charge distributions can be compensatedby the depth of the filters. However, such products cannot readily beapplied to delicate ion exchange processes. The number of active sites,as well as the accessibility of the active sites, is critical to thecapacity of such process. The chemical functional groups in ionexchangers cannot be buried close to the surface, but have to besomewhat removed from the surface, possibly with a molecular side armfor accessibility. One way of achieving this has been through theincorporation into the fibrous matrix of silanes which are chemicallymodified. Such silanes may carry functional groups such as DEAE, CM oraffinity chromatography sites. They are mechanically stable and strongand do not swell. However, they are expensive, and show very highnonspecific adsorption of protein by the silica hydroxy groups.

In sum, neither the ion exchange nor affinity chromatography supportscommonly used in laboratory scale purifications, nor the particulate (orion exchange modified particulate) containing fibrous matrices forchromatography or filtration have proven to be of great use in scale-upof delicate purification processes.

A need therefore continues to exist for supports useful in industrialscale ion exchange and affinity chromatography purification processes,which will be noncompressible, controllably swellable, have highexchange capacity, exhibit high flow rates, be versatile and berelatively inexpensive to produce. Recognizing this need, Hou et al.developed the invention embodied in the aforementioned application Ser.Nos. 466,114 and 576,448.

Purification of protein from bacteria contamination remains arecalcitrant problem in bioprocess. Hou et al, U.S. Pat. No. 4,361,486,discloses a bacteriocidal filter media which comprises an amount ofmetal peroxide immobilized in a substantially inert porous matrix. Bothgram-positive and gram-negative bacteria are negatively charged, mainlyowing to an excess of carboxyl and phosphate groups. Gram-positivebacteria contain both teichoic and teichuronic acids in their walls,whereas gram-negative organisms have phospholipids, with the negativelycharged lipid portion of lipopolysaccharides as components of theirouter membranes. In aqueous environments, the cell membrane exists as acontinuum of lipid and protein, organized as a molecular double layer,with the hydrophobic portions of the lipid molecule being opposed andthe hydrophilic groups projecting outwardly into the aqueous phase.

Phosphoglycerides account for about half the lipids, with polar groups,such as glycerol, serine, and carboxyl providing the hydrophiliccomponents. While various forms of proteins are embedded in the lipid,the major determinants of charge are surface polysaccharides covalentlylinked to the membrane proteins and lipids.

The effectiveness of bacteria removal through charge interaction hasbeen previously demonstrated, for example, by Ostreicher et al., U.S.Pat. Nos. 4,305,782 and 4,473,474 and Barnes, U.S. Pat. No. 4,473,475.Capture of bacteria, endotoxins, and viruses by charge modified filtersare described in Applied and Environmental Microbiology, 40: 892-896(1980). Positively charged ion exchange resins have been utilized forbacteria adsorption (Daniels, S. L., Development and IndustrialMicrobiology, 13: 211-253 (1972)).

Olson et al, U.S. Pat. No. 4,411,795 describes a variety of polymersattached to substrates including cellulose and recognized that thecombined effect of hydrophobic and ionic binding enhances adsorption oflipnin-containing cells.

Zvaginstev, D. G. et al., Mikrobiologiya 40: 123-126 (1971), concludedthat adsorption of bacterial cells by ion exchange resins wasattributable to electrostatic attraction between quaternary ammoniumgroups on the resin surface and carboxyl groups on the bacteria cellsurface. Hogg, in his Ph.D. thesis for the University of Salford,England (1976), demonstrated the interaction of bacteria withcellulose-based DEAE. The adsorption of several gram-negative organismswas shown, including Escherichia coli, Salmonella typhimurium, andPseudomonas aeruginosa.

The major forces impacting on bacterial adhesion to solid surfaces havebeen summarized by Rutter, P. R. in "The Physical Chemistry of theAdhesive of Bacteria and Other Cells," (1980) Microbial Adhesion toSurfaces, Editors Berkley et al., Ellis Howard Ltd., Publishers, WestSussex, England. According to Rutter, the Van der Walls force and chargeinteraction may be considered as long range forces. Where the distancebetween bacteria and solid surfaces are short, other interactions mustbe taken into account, for example, ion-dipole, dipole-dipole, hydrogenbonding, etc. The short range effects are particularly important inaqueous systems. When the bacteria particles approach the microscopicsolid surface, the local ordered water structure near the surface mustbe broken down. This leads to a short range repulsion force, which maybe sufficient to prevent the bacteria from coming closer to the solidsurface. On the other hand, when both the surfaces involved arehydrophobic, the short range interaction is a net attraction. Thisenergy favorable process, called "hydrophobic interaction", is the basisfor the well-known high performance liquid chromatography applied inprotein separations. As is known, a hydrocarbon chain of optimum length,when attached covalently to a solid matrix, may adsorb one protein inpreference to another due to the difference in hydrophobicity betweenproteins.

However, an overly strong hydrophobic solid surface may uncoil theprotein structure leading to the exposure of hydrophobic regions andincrease tendency for hydrophobic interaction. If the uncoiling is tooextensive, denaturation of the protein may result.

In most of the practical applications for bacteria and endotoxininactivation and removal from biological and pharmaceutical products,protein contamination by bacteria is the most prevalent problem. Onemust be able to inactivate and remove the microorganism-originatedcontaminants from protein specifically without causing loss ordenaturation of the final products. Accordingly, an optimal solid matrixshould exhibit a hydrophobic force which just matches the surfacehydrophobicity of proteins and maximally exploits these selecteddifferences in such hydrophobicity.

Thus, a need has continued to exist for a solid matrix for removal ofmicroorganism-originated contaminants from biological and pharmaceuticalproducts which will effectively eliminate the contaminants withoutdenaturing the final product.

Poly-quaternary ammonium polymeric polyelectrolytes are known to theprior art, these polymeric compositions produced by the polymerizationof a dihalide and a ditertiary amine. These polymers are characterizedby high charge density and have found substantial utility as flocculantsin the clarification of residential and industrial water supplies, ascatalysts in pigment retention additives, and as geling agents. Thesepolyeletrolyte materials are also known to be useful in the rheologicalmodification of fluids such as friction reducers, as dispersants forclay and sludge in both aqueous and oil-based systems, as anti-staticagents, and as additives to cosmetics, textile finishes and lubricatingoils. The materials are known to exhibit germicidal action or effectivebactericidal and fungicidal agents. See Rembaum et al., U.S. Pat. No.3,898,188.

Buckman et al., U.S. Pat. No. 3,784,649, discloses "high molecularweight" ionene polymeric compositions for utility, among others, asbroad spectrum microbicides for efficient control of bacteria includingsulphate reducers, fungi, algae, and yeast. The Buckman et al.polyionenes are suggested as additives to paper making systems, thepolyionenes increasing production per unit of equipment, improvingformation and strength properties of paper and paper board, andalleviating water pollution problems.

Rembaum, U.S. Pat. No. 4,046,750, discloses ionene modified beads foruse in binding small and large anionic compounds. The bead substratesare formed by the aqueous copolymerization of a substituted acrylicmonomer and a cross-linking agent. The formed polymeric beads arereacted with a mixture of a ditertiary amine and a dihalide or with adimethylaminoalkyl halide to attach ionene segments to the halo ortertiary amine centers on the beads. The thus-formed polyionene-modifiedbeads find use in affinity or pellicular chromatography for removal ofheparin from its mixture with polycations or neutral substances such asproteins or serums. Further disclosed utilities include use of themodified beads in the separation of cholesterol precursors such as bileacid from bile micellar suspensions, for binding RNA or DNAirreversibly, and a variety of other utilities which depend upon thebinding characteristics of the polycationic nature of the polyionene.

Rembaum, U.S. Pat. No. 4,013,507, discloses ionene polymers which bindnegatively charged mammalian cells such as malignant cells forselectively inhibiting the growth in vitro thereof. Conversely, U.S.Pat. No. 3,910,819 to Rembaum et al. discloses the use ofpolyionene-coated containers for increasing the rate of cell growth.

U.S. Pat. No. 3,927,242 to Rembaum et al. discloses the use ofpolyionenes as coatings for paper substrates. Further disclosed aresubstrates coated with the polyelectrolyte to maximize the bactericidalactivity of the polyionene. Suggested utilities include the impregnationof gauze material to form an antiseptic coagulant, germicidal dressingmaterial.

U.S. Pat. No. 4,075,136 to Schaper discloses a class of ionene polymerswhich contain certain functional groups such as nitriles, acrylates,vinyl acetates, ketones, acrolein, acrylamides, methosulfates, sulfonicacids, pyridines, and pyrrolidones. A host of utilities are disclosed,including the use of the functional ionene polymers as biocides and asfunctional coatings on paper, for example, electroconductive, adhesiveand photosensitive coatings.

In summary, polyionenes have been known and used for a substantialperiod of time and for a variety of purposes. However, the combinationof polyionenes with the modified polysaccharide substrates of thepresent invention have not been suggested.

SUMMARY OF THE INVENTION

It is therefore an object of the invention to provide a novel molecularsupport.

Another object of the invention is to provide a molecular support usefulfor ion exchange chromatography, affinity chromatography or reversephase chromatography.

Yet another object of the invention is to provide a chromatographicsupport useful for industrial scale chromatographic operations.

Still another object of the invention is to provide industrial processesof ion exchange, affinity chromatography, and reverse phasechromatography.

Yet another object of the invention is to provide processes for thepreparation of ion exchange, affinity and reverse phase chromatographicsupports.

This invention comprises a novel molecular support useful for ionexchange chromatography, affinity chromatography or reverse phasechromatography. The novel separation matrix provides chromatographicseparation useful for industrial scale chromatographic operations,industrial processes of ion exchange, affinity chromatography, andreverse phase chromatography. This invention further comprises processesfor the preparation of ion exchange, affinity and reverse phaseseparation matrices.

More specifically, the invention comprises a polyionene-transformedmodified polysaccharide separation matrix, the modified polysaccharidecomprising an insoluble polysaccharide covalently bonded to a syntheticpolymer, said synthetic polymer made from (a) a polymerizable compoundwhich has a chemical group capable of being covalently coupled directlyor indirectly to said polysaccharide; and (b) one or more polymerizationcompounds containing (i) an ionizable chemical group, (ii) a chemicalgroup capable of transformation to an ionizable chemical group, (iii) achemical group capable of causing the covalent coupling of saidpolymerizable compound (b) to an affinity ligand or to a biologicallyactive molecule, or (iv) a hydrophobic chemical group. Polymerizablecompounds (a) and (b) may be the same or different. The thus-modifiedpolysaccharide is then transformed by reactively bonding a polyionenethereto. The invention also comprises a process for preparing thepolyionene-transformed modified polysaccharide, the process comprisingpolymerizing the monomers, grafting the polymerized monomers to thepolysaccharide, and then reactively bonding the modified polysaccharidewith polyionene. Separation matrices derived from the aforementionedpolysaccharide materials are capable of acting as chromatographicsupports for ion exchange chromatography, for affinity chromatography,reverse phase chromatography or as reagents for biochemical reactors.The thus-transformed modified polysaccharide demonstrates a two-phaseadsorptive capability as a result of charge interaction with themicroorganism-originated contaminant as well as hydrophobic interactiontherewith. Additionally, the quaternary ammonium groups of thepolyionene are biocidal.

The polyionene-transformed modified polysaccharide will, in addition,demonstrate biocidal and bacterial adsorption capabilities.

In order to put the theoretical aspects of protein interaction phenomenainto practical use in a form of a separation device, a separation matrixwas developed by taking the following steps:

1. formation of a hydrophilic physical structure with controlled highporosity as backbone material;

2. synthesis of a reagent for adsorption of contaminants ofmicroorganism origin;

3. coupling or coating the reagent formed in step 2 to the physicalstructure of step 1.

The development of such separation matrices basically requires aphysical structure of sufficient rigidity to sustain the high liquidflow without causing structural deformation, yet the pore has to besufficiently large to accommodate the migration of large proteinmolecules without restriction. It must also be compatible with proteinsyet chemically stable under different aqueous conditions. The inventionof a matrix physical structure fulfilling such requirements has beenfully disclosed in our previously filed pending application Ser. No.576,448. The present invention provides an improvement thereon whereinthe modified polysaccharide is transformed by reactively bonding theretoa polyionene polymer.

These separation matrices have been attained by providing:

A modified polysaccharide material, which comprises:

1. a polysaccharide covalently bonded to a synthetic polymer;

2. said synthetic polymer made from at least one of

(a) a polymerizable compound which has a chemical group capable of beingcovalently coupled directly or indirectly to said polysaccharide; and

(b) one or more polymerizable compounds containing (i) an ionizablechemical group, (ii) a chemical group capable of transformation to anionizable chemical group, (iii) a chemical group capable of causing thecovalent coupling of said polymerizable compound (b) to an affinityligand or to a biologically active molecule, or (iv) a hydrophobicchemical group;

3. said modified polysaccharide having bonded thereto a polyionene.

Molecular separation materials derived from the aforementionedpolysaccharide materials are capable of acting as chromatographicsupports for ion exchange chromatography, for affinity chromatography,reverse phase chromatography or as reagents for biochemical reactors,wherein control of microorganisms-originated contaminants is essential.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will become better understood by reference to thedetailed description provided hereinafter when considered together withthe accompanying drawings, wherein

FIG. 1 is a diagram showing (A) an anion exchange cellulose derivatizedby a prior art approach which yields one cationic site per saccharideunit and (B) an anion exchange cellulose derivatized by the approach ofthe invention which yields multiple cationic sites per saccharide unit.

FIG. 2 shows the derivatized anion exchange cellulose of FIG. 1(B) afterfurther cross-linking or quaternization.

FIG. 3 shows the separation of gamma globulin type I (A), gamma globulintype II (B), and albumin (C), as per Example 10.

FIG. 4 shows results obtained in the elution of bound transferrin by pHshift using a DEAE cartridge, as per Example 11.

FIG. 5 shows the adsorption capacities for bovine IgG at various pH's asper Example 13.

FIG. 6 shows plasma fractionation using a quaternized cartridge mediumas per Example 14.

FIG. 7 is a partial sectional view of a side elevation of one embodimentof the chromatography column of this invention;

FIG. 8 is an enlarged cross-sectional view taken along line 2--2 of FIG.7;

FIG. 9 is a perspective view of the core with a portion of the solidstationary phase broken away therefrom showing the spirally woundchromatographic media and spacer means therebetween.

FIG. 10 is a cross-sectional view of another embodiment of the inventionwherein the chromatography column is in disc configuration.

FIG. 11 is a top plan view of the inlet housing member of the inventionembodiment in disc configuration.

FIG. 12 is a top plan view of the outlet housing member of the inventionembodiment in disc configuration.

FIG. 13 is a top plan view of one embodiment of the stationary phase ofthe invention column in disc configuration.

FIG. 14 is a side elevation of one embodiment of the stationary phase ofthe invention column in disc configuration.

FIG. 15 is a cross-sectional view of one embodiment of the stationaryphase of the invention column in disc configuration depicting aplurality of layers of separation media and spacer means interposedbetween adjacent layers of said separation media, prior to the sonicwelding of the peripheral edges.

FIG. 16 is a cross-sectional view of a preferred configuration for theinvention column in disc configuration. In this configuration, thehousing in disc configuration forms a radially outwardly expandingchamber. A portion of the spacer means is removed for clarity.

FIG. 17 is a graph demonstrating the effectiveness ofpolyionene-transformed cellulose at separation of Salmonella G-130 froma liquid.

FIG. 18 is a graph demonstrating the effectiveness ofpolyionene-transformed modified cellulose at separation of SalmonellaG-130 from biological fluids containing albumin.

FIG. 19 is a graph demonstrating the impact of column length on removalof bacteria from a liquid.

BRIEF DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is related to the discovery and development ofmaterials useful as insoluble supports for a variety of applicationsincluding a wide range of chromatographic separations, i.e., affinityseparations, or even as insoluble supports for bioreactors.

The support materials are based on a composite of an organic syntheticpolymer and a polysaccharide which is subsequently transformed bybonding polyionene thereto. In its most used embodiment the compositeper se is biologically inert. The organic synthetic polymer carrieschemical groups which are capable of coupling to said polysaccharide,and also carries chemical groups which can provide ion exchangecapacity, which can provide ion exchange capacity, which can provideanchoring capacity for affinity ligands or for biologically activemolecules in general, or which can provide hydrophobic groups.

Strictly speaking, the polymer coupled to the polysaccharide may beeither a copolymer or a homopolymer. When the chemical groups capable ofcoupling to the polysaccharide are the same as the chemical groupsuseful as anchoring units for affinity ligands or biologically activemolecules, the polymer, in this particularly form would be ahomopolymer. In another form, however, the polymer is a copolymercontaining groups capable of coupling to the polysaccharide and alsodifferent groups capable of serving as anchoring groups for molecules.

The invention also relates to ion exchange materials obtained from thepolyionene-transformed modified polysaccharide which are obtained byfurther reaction with crosslinking agents, and/or by further reactionssuch as chemical ionization, or unmasking of potentially masked ionexchange groups.

The invention also relates to materials derived from thepolyionene-transformed modified polysaccharide by attaching theretoaffinity ligands or biomolecules, to thereby obtain affinitychromatography or bioreactive materials, or attaching theretohydrophobic substituents for reverse phase chromatography.

The invention also relates to mixtures of the aforementioned materialswith unmodified polysaccharides, with modified or unmodified particulatematerial, or mixtures thereof to give a variety of separation media.

MATERIALS

The term "polysaccharide" as used in the specification and claims ismeant to include compounds made up of many--hundreds or eventhousands--monosaccharide units per molecule. These units are heldtogether by glycoside linkages. Their molecular weights are normallyhigher than about 5,000 and up into the millions of daltons. They arenormally naturally occurring polymers, such as, for example, starch,glycogen, cellulose, gum arabic, agar and chitin. The polysaccharideshould have one or more reactive hydroxy groups. It may be straight orbranched chain. The most useful of the polysaccharides for the purposesof this invention is cellulose.

The polysaccharide is preferably fully unprotected and carries all ofits hydroxy groups in the free state. Some blocking of the hydroxygroups is possible, as for example by acylation or aminoacylation.Extensive blocking of the hydroxy groups of the polysaccharide, however,is undesirable since the polysaccharide thereby loses its hydrophiliccharacter, which is necessary to provide appropriate chemicallycompatible interaction with biomolecules. If the polysaccharide becomestoo hydrophobic, negative interactions with such molecules as proteinsleads to possible non-specific bonding and denaturation phenomena. Also,if the masking of the polysaccharide hydroxy groups is too extensive,the reactivity of the resulting material with the polymer is greatlydiminished. For all of these reasons, it is preferred to retainsubstantially all hydroxy groups in the free state. The polysaccharidemay, however, be chemically activated, as seen infra.

Cellulose is the preferred polysaccharide. By "cellulose" is intended tomean any of the convenient and commercially available forms of cellulosesuch as wood pulp, cotton, hemp, ramie, or regenerated forms such asrayon. There exists no criticality as to the selection of a suitableform of cellulose. Cellulose is a naturally occurring polysaccharideconsisting of beta -1,4- linked glucose units. In the native state,adjacent cellulose chains are extensively hydrogen bonded formingmicrocrystalline regions. These regions are interspersed by amorphousregions with less hydrogen-bonding. Limited acid hydrolysis results inpreferential loss of the amorphous regions and gives so-calledmicrocrystalline cellulose. The cellulose useful in the presentinvention is either cellulose in the native state, or in themicrocrystalline state. Also, cellulose derived from cotton linter isbetter than that derived from wood pulp, as the latter contains lignin.

Chemical reactions to attach the polymer to the polysaccharide materialnormally proceed with difficulty in crystalline regions but take placemore readily in amorphous regions. For example, the substitution offunctional groups into cellulose has a disruptive effect on thestructure thereof. If carried out to completion, the cellulose matrixwould be destroyed and utimately water soluble polymers would be formed.Typical examples of this phenomenon are the hydroxyethyl cellulose andcellulose gums of the prior art, which become the commonly usedadhesives and binders after dissolving in water.

Each anhydrous saccharide unit in a polysaccharide molecule may havethree or more reactive hydroxy groups. Theoretically, all three or morecan be substituted with the polymer. The product from such reaction,however, would have a degree of substitution of three or more, which inthe case of ion exchange materials, would render it soluble. Even atlevels of substitution below those at which total water solubilityoccurs, such polysaccharide derivatives become unsuitable aschromatographic supports. Therefore, substitution of the polysaccharideis restricted to the more reactive centers of the amorphous regions andis seldom carried out beyond the level of about 1 meq/gm of dry weightin fiber form. At this level of substitution, the native configurationof the polysaccharide structure is only slightly modified, and the lowdensity non-uniform exchange sites are readily accessible to largebiomolecules.

The final structure of a molecular support of the invention thuscomprises a polysaccharide chain covalently modified at a multiplicityof sites along such chain with the synthetic polymers.

The polymer which modifies the polysaccharide is either a homopolymer ora copolymer. The definition of the polymer as a homo- or copolymerdepends on whether the polymerizable compounds (a) and (b) aredifferent. In its most general form, the copolymer could be a random, ablock or an alternating copolymer.

In one embodiment, the polymerizable compound (a) (also called"comonomer (a)") may have a group capable of reacting with a hydroxygroup of polysaccharide with the formation of a covalent bond. Suchpolymerizable compounds are defined for example in U.S. Pat. No.4,070,348 to Kraemer et al., which is herein incorporated by reference.The chemical groups are capable of reacting with hydroxy groups attemperatures up to those at which the polysaccharide begins to decomposeor depolymerize, e.g., 0° to 120° C., in aqueous solution and therebyform covalent bonds with the oxygen atoms of the hydroxy groups. Sincewater is always present in considerable excess with respect to thehydroxy groups, chemical groups which react spontaneously with water,such as, for example, isocyanate groups, are less suitable. Aqueoussolutions comprise pure water or mixtures of water with one or morewater miscible co-solvents, such as alcohols, ketones, and the like.

Hydroxy reactive groups of comonomer (a) are preferably activatedcarboxy groups such as are known from peptide chemistry or O-alkylatingagents, such as alkyl halide or epoxide groups. Representatives of theO-alkylating comonomers are acrylic- and methacrylic anhydrides,acrylolylmethacryloyl N-hydroxy succinimides, omega-iodo-alkyl esters ofacrylic or methacrylic acid in which the alkyl group in general contains2 to 6 carbon atoms, allyl chloride, chloromethylstyrene, chloroacetoxyethyl methacrylate, and compounds having a glycidyl group. The latterare ethers or esters formed between a glycidyl alcohol and anunsaturated alcohol or unsaturated carboxylic acid, respectively. Theglycidyl alcohols are aliphatic and cycloaliphatic alcohols and etheralcohols having from 3 to 18 carbon atoms which are esterified with analpha,beta-unsaturated carboxylic acid, preferably acrylic ormethacrylic acid, or are etherified with an olefinically oracetylenically unsaturated alcohol. Typical compounds are glycidylacrylate and methacrylate; 4,5-epoxy-pentylacrylate;4-(2,3-epoxy-propyl)-N-butyl-methacrylate; 9,10-epoxy-stearylacrylate;4-(2,3-epoxypropyl)-cyclohexyl methacrylate; ethyleneglycol-monoglycidyl etheracrylate; and allyl glycidyl ether.

Preferred monomers for the homopolymers or copolymers of the syntheticpolymer grafted to the polysaccharide are glycidyl acrylate and glycidlymethacrylate, with a homopolymer of glycidyl methacrylate mostpreferred. Such homopolymers may be further modified by conversion tocarboxylic groups (by reaction with, for example, acrylic acid) toamine-containing groups (by reaction with, for example, a diamine) andto hydroxy groups.

If the active comonomer units (a) are sensitive to hydroxy groups, andif they do not react with the polysaccharide offered, they may betransformed, in the presence of water, into hydrophilic carboxy orhydroxy groups. The activated groups are therefore present in thepolymeric material in generally greater number than is necessary for thebonding with the polysaccharide.

In another embodiment, the polymerizable compound (a) may be one whichdoes not react directly with hydroxy groups of the polysaccharide, butrather is covalently coupled to the polysaccharide indirectly, via abridge compound. This is the case when the polysaccharide is firstchemically activated as by oxidation, and reacted with a compoundhaving, e.g., an epoxy group or a vinyl group, capable of reaction withan appropriate functionality of polymerizable comonomer (a).

The polymerizable comonomer (b) will vary depending on the ultimate useof the carrier material. If the carrier material's ultimate use is toserve as an ion exchange chromatographic material, the comonomer (b) maycontain any of the well known ionizable chemical groups or precursorsthereof such as compounds containing a vinyl or vinylidine group and acarboxylic acid, a carboxylate salt, a carboxylate ester, preferablyhaving 1 to 6 carbon atoms, a carboxylic acid amide, a secondary or atertiary amine, a quaternary ammonium, a sulfonic acid, a sulfonic acidester, a sulfonamide, a phosphoric or phosphonic acid, or aphosphoramide or phosphonamide group.

When comonomer (b) carries the precursor of a material having ionexchange properties, the ion exchangable group itself can be obtained byunmasking, such as for example, by selective hydrolysis of an anhydride,ester or amide, or salt formation with an appropriate mono-, di- ortrivalent alkaline or alkaline earth metal, as is otherwise well-knownin the art.

Preferred ion exchange functionalities for comonomer (b) are aminoethyl,carboxymethyl, carboxyethyl, citrate, diethylaminoethyl, ecteola (mixedamines), guanido ethyl, phosphonic acid, p-aminobenzyl, polyethyleneimine, sulphoethyl, sulphomethyl, triethylaminoethyl, or chelatinggroups such as --N(CH₂ CO₂ H)₂. When the ultimate use of the carriermaterial is as a support for an affinity liquid, comonomer (b) carries achemical group capable of causing the covalent coupline of saidcomonomer (b) to an affinity ligand, i.e. an "anchoring" group. Sincemost affinity ligands carry nucleophiles such as hydroxy, amino, thiol,carboxylate, and the like, any electrophilic group capable of reactingwith such nucleophile can be present in comonomer (b). Suchelectrophilic groups include, but are not limited to, those describedpreviously as active groups capable of reacting with the hydroxy groupof cellulose. They also include activated carboxy groups used in peptidechemistry for the formulation of peptide bonds, such as carbonylchlorides, carboxylic anhydrides and carboxylic acid azide groups, aswell as phenyl esters and aldehydes used for the formation of Schiff(imine) bases.

Also useful are the carboxylates of hydroxylamino derivatives of theformula (1) ##STR1## in which R is an alpha,beta-unsaturated,polymerizable radical and R' and R" are identical or different C₁ -C₆alkyl or alkanoyl groups. R'" may be a direct bond (--) or a C₂ -C₃alkyl group. R' and R" together with the N atom may also form aheterocyclic ring. Typical compounds of this type are: ##STR2##

Other compounds having activated carboxyl groups include acryloyl- andmethacryloyl chloride, acrylic and methacrylic anhydride, maleicanhydride, phenyl acrylate and methacrylate, glycidyl acrylate andmethacrylate, and 4-iodobutylacrylate and methacrylate.

A very useful potentially electrophilic reactive group in comonomer (b)useful for coupling to an affinity ligand is a group capable of beingactivated to an electrophilic group with a reagent such as a cyanogenhalide. It is known in the art the cyanogen halides react with 1,2-diolsto yield activated structures of the following types: ##STR3##

This structure is then capable of reacting with the nucleophile of anaffinity ligand. Among the preferred 1,2-diols present in comonomer (b)are various saccharides, including monosaccharides such as glucose,mannose and galactose, disaccharides such as lactose and maltose,trisaccharides such as raffinose or, generally, glycosides. The1,2-diol-containing functional group can be attached to thepolymerizable comonomer (b) by such reactions as esterification, amideformation, and the like. Among the most preferred of these is thereaction of glycidyl acrylate or methacrylate with a saccharide, toyield an ether-containing comonomer (b).

When the ultimate use of the carrier material is as a carrier forbiological molecules, any of the anchoring groups mentioned forcomonomers (a) or (b) can also be used. Other types of activated groupssuch as those containing aldehydes or amines can also be used.

The polymerizable comonomer (b) can be substantially of one type or canbe a mixture of one or more types. This is particularly applicable whenthe ultimate use of the material is as an ion exchange carrier.Comonomers (b) can then contain such functional groups as anionicexchange groups and cationic exchange groups in various differentratios, if desired.

Preferably, the polymerizable monounsaturated compounds (b) arepolymerizable compounds of the formula (4): ##STR4## wherein R¹ ishydrogen or methyl;

A is CO, or SO₂ ;

X is OH, OM (where is a metal ion), OR² (where R² is a straight orbranched chain C₁ -C₁₈ alkyl group), OR³ OH (where R³ is a straight orbranched chain C₂ -C₆, alkyl or aromatic group), NR⁴ R⁵ or N⁺ R⁴ R⁵ R⁶(where R⁴ is the same or different as R⁵ which is the same or differentas R₆, and are hydrogen, R² or R³ OH);

AX may also have formula (5): ##STR5## wherein Y is --CO₂ ⁻, --CH₂ CO₂O⁻, --SO₃ ⁻, --CH₂ SO₃ ⁻, --PO₄ H⁻, --CH₂ PO₄ H⁻, --CH₂ N(CH₂ --COO⁻)₂,--CH₂ --NR⁴ R⁵, or --CH₂ --NR⁴ R⁵ R⁶, or the corresponding free acid,ester or partial ester groups, as described previously. In theseformulae, the groups R⁴, R⁵ ; R⁴, R⁶ ; or R⁵, R⁶ may form a 5-7 memberedheterocyclic ring with the nitrogen atom. R⁴, R⁵, and R⁶ are aspreviously defined.

Alternatively, (and when the material is to be used as an anchor foraffinity ligands or biomolecules), A is CO or SO₂, and X is mostpreferably O--CH₂ --CH(OH)--CH₂ -Saccharide, where "-Saccharide" is amono-, di- or polysaccharide having a group which can be activated forreaction with nucleophilic reactive groups on the affinity ligand or thebiomolecule by a cyanogen halide.

The preferred comonomer (a) for anionic exchange materials is glycidylacrylate or methacrylate. The preferred comonomer (b) for anionicexchange materials is diethylaminoethyl acrylate or methacrylate. Themost preferred comonomer (b) for anchoring materials is the comonomerobtained from reaction of glycidyl acrylate or methacrylate withglucose.

The preferred comonomer (a) for cationic exchange materials isaminoethyl methacrylate, coupled to the polysaccharide by previousoxidation thereof. The preferred comonomer (b) for cationic exchangematerials is methacrylic acid, acrylic acid and acrylic acid dimer, orglycidyl methacrylate further oxidized to a carboxylic acid group aftercopolymerization.

However, as stated above, where the primary function of the matrix isfor removal of microrganism-originated contaminants, the preferredmodifier for the polysaccharide is a homopolymer of glycidylmethacrylate, perhaps reacted to provide carboxy, amine or hydroxyreactive sites.

The average molecular weight of the polysaccharide-modifying polymer isdependent on the number of monomers present therein. It is required tohave at least a sufficient number of comonomers (a) so as to be able toform covalent attachment throughout amorphous regions of thepolysaccharide surface. The number of comonomers (b) cannot be toosmall, since otherwise the exchange capacity, or theanchoring/interacting capacity is negligible. The number of comonomers(b) can neither be too high, since this would cause great difficulty inthe reaction between the reactive groups of comonomer (a) and thepolysaccharide. Preferably, the polysaccharide-modifying copolymercarries anywhere between 1 and 500 units (a) plus (b), most preferablybetween 20 and 100 units. This corresponds to molecular weights ofbetween about 100 and 100,000, preferably between 1,000 and 10,000.

Other neutral comonomers (c), different than those represented by (i),(ii), (iii) or (iv) supra, can also be added to the polymer, if desired.These comonomers may be polymerizable unsaturated compounds carryingneutral chemical groups such as hydroxy groups, amide groups, alkylgroups, aryl groups and the like. Preferred among comonomers (c) are C₁-C₆ alkyl acrylates or methacrylates, or the corresponding hydroxy alkylacrylates or alkacrylates. The function of comonomers (c) may be toincrease the presence of hydrophobic or hydrophilic residues in thepolymers, so as to provide a desired balance of hydrophilic andhydrophobic groups, if necessary.

The minimum ratio of comonomer (a) to total comonomer content isimportant. The synthetic polymer should have a sufficient amount ofcomonomer (a) to permit substantial covalent coupling of the polymer tothe polysaccharide. If too little comonomer (a) is present in thepolymer, then grafting becomes difficult, if not impossible. Generally,a minimum of about 4-12, preferably 5-10% by weight of comonomer (a)relative to the total of (a) plus (b) (and (c) if any is present) isneeded. Amounts of about 0.5 to 1 or 2% by weight appear to merelycross-link the polymer without substantial grafting onto thepolysaccharide.

The upper limit of comonomer (a) in the polymer can be varied up to99.9% by weight depending on the desired amount of rigidity,functionality and hydrophilicity. Increasing the amount of comonomer (a)too much above 15 to 20% by weight, however, decreases the porosity.Large molecules then have difficulty in graining full access to thefunctional groups in comonomer (b). Of course, wheremicroorganism-originated contaminants are the primary concern, this isnot a factor. In some instances, it may be preferred to have apredominance of comonomers (b) over comonomers (a). Comonomers (c) maybe present in an amount of up to 20 percent by weight of the total (a)plus (b) plus (c).

The weight ratio of polysaccharide to the modifying polymer is freelyadjustable, and varies from 0.1 to 5 weight parts of copolymer to partsby weight of the polysaccharide.

When comonomers (b) carry ionizable chemical groups capable of providingcation exchange capacity, it is found that unless some degree ofcrosslinking is provided, the flexibility of the material in solutiontends to favor the formation of micelle-type aggregates and slow loss ofcapacity. Therefore, it is a preferred mode of the invention to providepolymeric crosslinking for these types of modified polysaccharides.Crosslinking can be provided either by incorporating into thepolymerization recipe a small amount of polyunsaturated comonomer havingat least two polymerizable alpha,beta-carbon double bonds, such as forexample mono- and polyethylene glycol dimethacrylates and diacrylates(with the polyethylene glycol residue containing up to 6 ethylenegroups), ethylene dimethacrylate, ethylene diacrylate, tetramethylenedimethacrylate, tetraethylene diacrylate, divinylbenzene, triallylcyanurate, methylene-bis-acrylamide or -bis-methacrylamide, and thelike.

Another type of crosslinking agent is particularly applicable tocopolymers made from an aminoalkyl comonomer (b). Because of thepresence of a free pair of electrons on the aminoalkyl nitrogen atoms,crosslinking can be carried out with such bifunctional reagents as wouldreact with nitrogen free electron pairs. Among these are the diacylhalides, such as Hal--CO--(CH₂)_(n) --CO--Hal, or the alkyl dihalides,such as Hal--(CH₂)_(n) --Hal, wherein Hal is a halide such as chloride,bromide or iodide, and n may be anywhere between 2 and 12. Otherbifunctional reagents capable of reaction with nitrogen atoms can alsobe used. The advantage of these bifunctional reagents is that theysimultaneously crosslink the copolymer, while also providing a cationiccharge at the nitrogen centers, thereby ionizing the material.

The amount of crosslinking agent is best determined empirically. It isto be considered sufficient when the polymer preserves the ion exchangecapacity at a constant value over time, yet would be too high ifswelling is prevented, and too much rigidity is obtained in the finalmaterials. Ideally, an amount of crosslinking agent between 5 to 20 molepercent of the synthetic polymer units is sufficient.

By the term "affinity ligand" as used throughout the present applicationand in the claims, is meant to include any small or high molecularweight molecule which can be immobilized in a stationary phase and usedto purify a complementary binding molecule from a solute phase byaffinity chromatography. For example, a ligand can be an inhibitor, acofactor, a prosthetic group, or a polymeric substrate, all of theseuseful to purify enzymes or holoenzymes. Other ligand/ligate pairs areenzymes/polymeric inhibitors; nucleic acid, single strand/nucleic acid,complementary strand; hapten or antigen/antibody; antibody/proteins orpolysaccharides; monosaccharides or polysaccharides/lectins orreceptors; lectins/glycoproteins or receptors; small targetcompounds/binding proteins; and binding protein/small target compounds.When antigen/antibody pairs are used as the ligand/ligate pair, thetechnique takes the particular name "immunoaffinity" chromatography.

The "biologically active molecule" which can be bound to the carriers ofthe invention can include enzymes, enzyme substrates, inhibitors,hormones, antibiotics, antibodies, antigens, peptides, saccharides,nucleic acids, and the like. The only requirement for these molecules isthat they have reactive groups thereon which can be covalently coupledto the anchoring chemical groups on comonomer (b).

Of particular interest is the immobilization of enzymes such ashydrolases, isomerases, proteases, amylases, and the like. Theseimmobilized enzymes can then be used in biochemical reactors, as isotherwise well-known in the art.

By the use of the term "reverse phase chromatography" or "hydrophobicinteraction chromatography" is meant to include chromatography used toadsorb hydrophobic components in mixtures. Such components includelipids, cell fragments and the like. In this embodiment, comonomer(b)(iv) is usually an acrylate or methacrylate ester of C₆ -C₁₈ straightor branched chain alcohols, or of aromatic alcohols such as phenol ornaphthol.

However, in a preferred configuration wherein the modifiedpolysaccharide is to be utilized as a separation matrix, in combinationwith a polyionene, for the bacterial decontamination of pharmaceuticaland/or biological solutions, the polysaccharide is cellulose and themodifier thereof is a homopolymer of glycidyl methacrylate as in Example3 below.

The term "polyionene" or "ionene-type polymeric composition," firstcoined by Rembaum, A. et al., Polymer Letters, 6: 159-171 (1968), hasbeen adopted by other authors in the field, including ChemicalAbstracts, as reported in U.S. Pat. No. 3,784,649. However, for thepresent invention, "polyionene" is meant to include those water-solublepolymers having polyquaternary ammonium groups separated by hydrophobicgroups, said hydrophobic groups comprising aromatic groups or alkylgroups containing at least six carbon atoms.

The polyionenes of the present invention include those polymers havingthe following repeating units: ##STR6## wherein R⁷ and R⁸ are C₁ -C₄alkyl; L is --(CH₂)_(n) --P--(CH₂)_(m) ; M is --(CH₂)_(o) --Q--(CH₂)_(p)--, with P and Q being the same or different and representing at leastone of CH₂, CHA, C₆ H₄, pyridine, C₆ H₃ A, C₆ H₄ --CHA--C₆ H₄, or R⁹ C₆H₂ A; wherein A is a reactive group, typically OH, amino, aldehyde,halide, epoxy and carboxy, wherein R⁹ is C₁ -C₄ alkyl; and wherein m, n,o and p represent integers from 1 to 20.

In general, polyionene polymers are prepared by reacting a dihaloorganic compound with a secondary or tertiary amine. Typicalpolymerization processes and reactants are described in U.S. Pat. No.2,261,002 to Ritter; U.S. Pat. No. 2,271,378 to Searle; U.S. Pat. No.3,489,663 to Bayer et al., U.S. Pat. No. 3,784,649 to Buckman et al.;and U.S. Pat. No. 4,038,318 to Tai. Additionally, Rembaum, at U.S. Pat.Nos. 3,898,188; 3,910,819; 3,927,242; and 4,013,507 describes additionalpolyionenes and their synthetic procedures. Each of the above-referencedU.S. patents is specifically incorporated in its entirety herein.

Typical dihalo organic compounds included within the scope of thepresent polyionene precursors include those compounds having thefollowing general formula:

    X--(Y).sub.q --Z--(Y).sub.r --X

where X is I, Br, or Cl; Y represents a CH₂ group and/or a substitutedCH₂ group wherein one of the hydrogens is replaced with a C₁ -C₄ alkylor hydroxy-substituted C₁ -C₄ alkyl; and q and r independently representintegers varying from 1 to 10. Z represents CH₂, CHA, C₆ H₄, C₆ H₃ A, C₆H₄ --CHA--C₆ H₄ and R⁹ C₆ H₂ A where R⁹ represents a C₁ -C₄ alkyl, withA defined as above.

In a preferred embodiment, wherein the modified polysaccharide substrateis treated with the polyionene, Z represents a moiety such as CHA, C₆ H₃A or R⁹ C₆ H₂ A, the presence of the A group providing a linking sitefor subsequent reaction with the modified polysaccharide substrate.

Suitable secondary and ditertiary amines include, but are not limited toN,N,N',N'-tetramethylethylenediamine,N,N,N',N'-tetraethylethylenediamine,N,N,N',N'-tetramethyl-1,3-butanediamine,N,N,N',N'-tetraethyl-1,3-butanediamine,N,N,N',N'-tetramethyl-1,4-butanediamine,N,N,N',N'-tetraethyl-1,4-butanediamine, N,N'-dimethylpiperazine,N,N'-diethylpiperazine, 1,4-diazabicyclo(2,2,2)octane, 4,4'-dipyridyl,N,N,N',N'-tetramethylbenzidine, N,N,N',N'-tetraethylbenzidine,oxy-bis-2,2(N,N-dimethylethylamine),4,4'-bis(dimethylamino)benzophenone,p,p'-methylene-bis(N,N'-dimethylaniline),N,N,N',N'-tetrakis(hydroxyethyl)ethylenediamine,N,N,N',N'-tetrakis(2-hydroxypropyl)ethylenediamine,N,N,N',N'-tetramethyl-2-butenediamine,N,N,N',N'-tetramethyl-1,6-hexanediamine,bis(dimethylaminomethyl)benzenes, bis(dimethylaminomethyl)toluenes,bis(dimethylaminomethyl)xylenes, dimethylamine, diethylamine,diisopropylamine, dibutylamine, diethanolamine, diisopropanolamine,piperidine, morpholine, 2,6-dimethyl-morpholine,1,2,4-trimethylpiperazine, and1,4-bis(2-hydroxypropyl)-2-methylpiperazine.

Among the preferred reactants are the N,N,N',N'-tetra-(C₁ -C₄ alkyl)alkyl diamines having 1 to 10 carbon atoms between the substituted aminegroups. More preferred is N,N,N',N'-tetramethyl-1,6-hexanediamine andN,N,N',N'-tetraethyl-1,6-hexanediamine.

Included as well, however, are compounds having the general formula:

    N(CH.sub.3).sub.2 --CH.sub.2 --M--CH.sub.2 --N(CH.sub.3).sub.2

where M is a reactive moiety-containing group such as hydroxy, amino,aldehyde, carboxy, halide and epoxy. Typically, M maybe CHOH, C₆ H₃ OH,C₆ H₄ CHOHC₆ H₄ and R⁹ C₆ H₂ OH, with R⁹ as described above.

Typical reactions for the synthesis of the polyionenes for the practiceof the present invention wherein the polyionenes are coated or graftedonto the modified polysaccharide substrate are as follows: ##STR7##where n is a whole number integer. Typically, the polyionenes of thisinvention have molecular weights in the range of 10,000 to 100,000.

Where, for example, x is 6 and y is 6, the resulting product is a6,6-polyionene having the general formula: ##STR8## where x is 6 and yis 10, the resulting product is a 6,10-polyionene having the generalformula: ##STR9##

Also contemplated are the reaction products wherein a mixture of dihalocompounds are used, for example, where Cl--(CH₂)₆ --Cl and Cl--(CH₂)₁₀--Cl are reacted with a secondary or ditertiary diamine, yielding aproduct having the general formula: ##STR10## where m and n are wholenumber integers.

The last above concept is especially useful where it is desirable tosynthesize polyionenes which contain reactive groups, i.e. hydroxylgroups. A typical reaction is as follows: ##STR11##

Similarly, reactive group-containing polyionenes may be obtained fromthe following reaction: ##STR12## where Ra is, for example, a C₁ -C₄alkyl.

By reacting the pendant reactive group with, for example,epichlorohydrin, the polyionene may be restructured to contain epoxygroups, said epoxy groups themselves useful for bonding onto themodified polysaccharide substrate.

Another manner for creating reactive sites on the polyionene forsubsequent bonding is as follows: ##STR13##

The resulting polyionene, containing a substituted halide (chloride) inthe heterocyclic ring, may then be reacted with a suitable hydrophobicspacer arm, for example NH₂ --(CH₂)₃ --NH--(CH₂)₃ --NH₂, to yield areaction product with an amine terminated linker arm: ##STR14## saidamine terminated linker arm may subsequently be reacted with, forexample, epoxy functional groups on the modified polysaccharide tothereby graft the polyionene to the modified substrate.

An alternative procedure for creating reactive groups, i.e. hydroxygroups, on the polyionene is as follows: ##STR15##

This product may be reacted with dihalo organic compounds and ditertiarydiamines to produce a polyionene containing reactive hydroxyl groups.Reaction of that product with, for example, epichlorohydrin, results inepoxidation of the polyionene as above.

Another method for producing reactive group-containing polyionenes is asfollows: ##STR16##

Further, a mixture of ditertiary diamines and a reactivegroup-containing ditertiary diamine, for example,1,3-bis-(dimethylamino)-2-propanol may be reacted with a dihalo compoundas follows: ##STR17##

As above, the hydroxyl group may then be reacted with, for example,epichlorohydrin to produce an epoxidized derivative for bondingpurposes.

By the term "polyionene-transformed modified polysaccharide" is meant toinclude any and all of the above-described modified polysaccharideswhich have been transformed by the presence of a polyionene bonded tothe surface of said modified polysaccharide. In one embodiment, thepolyionene is reactive coupling to the modified polysaccharide throughthe hydroxy coupling groups thereon. The coupling process substantiallyimproves the resistance of the polyionene-transformed modifiedpolysaccharide to leaching during use thereof.

By the term "bonding" or "bonded" is intended that the polyionene issufficiently attached to the modified polysaccharide that saidpolyionene will not significantly be extracted under the intendedconditions of use.

By the term "microorganism-originated contaminants" or "contaminants ofmicroorganism origin" is meant to include bacteria, bacterial productssuch as endotoxins, viruses, micoplasma and the like.

By the term "biological liquids" is meant to include each and everyliquid system which is derived from or amenable to use with livingorganisms. Such liquids are ordinarily handled and processed undersanitary or sterile conditions and therefore require sanitized orsterilized media for separation. Included within such terms are isotonicsolutions for intramuscular or intravenous administration, solutionsdesignated for oral adminstration, solutions for topical use, biologicalwastes or other biological fluids which may comprise filterable bodiessuch as impurities, e.g. bacteria, viruses, or endotoxins which aredesirably isolated or separated for examination or disposable byimmobilization or fixation upon or entrapment within separation media.

Separation media in accordance with this invention may be employed aloneor combination with other separation media to treat pharmaceuticals suchas antibiotics, saline solutions, dextrose solutions, vaccines, bloodplasma, serums, sterile water or eye washes; beverages such as cordials,gin, vodka, beer, scotch, whiskey, sweet and dry wine, champagne orbrandy; cosmetics such as mouthwash, perfume, shampoo, hair tonic, facecream, or shaving lotion; food products such as vinegar, vegetable oils;chemicals such as antiseptics, insecticides, photographic solutions,electroplating solutions, cleaning compounds, solvent purification andlubrication oil; and the like, where retention of submicronic particles,removal of contaminants of microorganism origin, and resolution ofcolloidal hazes is desired. The separation media may be used to isolateblood parasites from peripheral blood and also to removemicroorganism-originated contaminants from peripheral blood. Includedamong the contemplated utilities are the use of the separation media forseparating contaminants from blood to be used for re-infusion. Ren, H.E. et al., Arch. Biochem. Biophys., 209: 579 (1981).

The carrier materials of the present invention can be used per se in thesame manner as other polysaccharide-based carrier materials of the priorart. Alternatively, and in a preferred mode, the polysaccharidematerial, which is preferably in fibrous form after the modification andpolyionene transformation, can be formed into a self-supporting fibrousmatrix, such as a fibrous sheet, with ion exchange properties, affinitychromatography properties, bioreactive or reverse phase properties,while at the same time demonstrating the capacity to removemicroorganism-originated contaminants. The modified fibrouspolysaccharide fibrous media can also incorporate unmodified fibers ofvarious different sizes, and, in addition, can also incorporate modifiedor unmodified particulate material. However, due to the biocidal andentrapping characteristics of the polyionene-transformed modifiedpolysaccharides, these separation media are especially well suited fordecontamination of biological and pharmaceutical solutions.

The fibrous media comprises a porous matrix of fiber wherein, because ofthe nature of the present invention, the fiber is effective formolecular or ionic separations or molecular reactions. The matrix issubstantially homogeneous with respect to each component. When aparticulate is present, it is preferred to modify it so that it is alsoeffective for molecular or ionic separations or reactions. Such aparticulate should be contained in the fibrous phase in an effectiveamount to achieve the desired separations or reactions. The overallmedia is substantially inert and dimensionally stable.

The preferred particulates which can be used include all of thosesubstances which can be provided in finely divided form and exhibitchromatographic functionality, i.e., capable of effective molecularseparations and/or reactions. Mixtures of such compositions may also beutilized. Exemplary of such particulates are silica, alumina, zirconiumoxide, diatomaceous earth, perlite, clays such as vermiculite, carbonsuch as activated carbon, modified polymer particulates such as otherion exchange resins, crystalline cellulose, molecular sieves, and thelike, the surfaces of which may be modified in a conventional manner.Such materials are commercially available under a variety of trademarkssuch as Biosila, Hi-Flosil, Li Chroprep Si, Micropak Si, Nucleosil,Partisil, Porasil, Spherosil, Zorbax cil, Corasil, Pallosil, Zipax,Bondapak, LiChrosorb, Hypersil, Zorbax, Perisorb, Factosil, CorningPorous Glass, Dowex, Amberlite resins, and the like.

Examples of references which describe particulates effective formolecular separations are Miller, U.S. Pat. No. 3,669,841, Kirkland etal., U.S. Pat. No. 3,722,181, Kirkland et al., U.S. Pat. No. 3,795,313,Regnier, U.S. Pat. No. 3,983,299, Chang, U.S. Pat. No. 4,029,583, Stehl,U.S. Pat. No. 3,664,967, Krekeler, U.S. Pat. No. 4,053,565 and Iher,U.S. Pat. No. 4,105,426. The entire disclosures of all of thesereferences are incorporated by reference herein.

The particle size of the particulate is not critical but influencessomewhat the flow rate at which the sample to be treated passes throughthe material. Usually, uniform particle sizes greater than about 5microns are preferred, with about 10-100 microns constituting apractical operational range. The amount of the particulate can varywidely from about 10 wt.% up to 80 wt.% or more of the solid phase. Theoptimum particulate concentration will vary depending on the molecularseparation desired.

The fibrous media should be capable of immobilizing the particulatecontained therein, i.e., capable of preventing significant particulateloss from the stationary phase, yet having a porosity which enables thefluid to pass through the media. Thus, although the modified cellulosematerials of the present invention are self-bonding and the addition ofextra fibers or binders may not be necessary, it is possible to utilizesuch extra fibers or binders. Other fibers usable for the media includepolyacrylonitrile fibers, nylon fibers, wool fibers, rayon fibers andpolyvinyl chloride fibers, other cellulose fibers such as wood pulp andcotton, and cellulose acetate.

One embodiment of the invention is the provision of a fibrous mediacomprising two different types of celluloses: one a modified celluloseaccording to the invention and another an unmodified cellulose.

Another embodiment of the invention, which may also be coupled with theaforementiond celluloses is an unrefined structural fiber which assistsin providing sheets of sufficient structural integrity in both the wet"as formed" condition, and in the final dry condition, and also allowshandling during processing as well as suitability for the intended enduse. Such fibers are typically relatively lrge, with commerciallyavailable diameters in the range of 6 to 60 micrometers. Wood pulp canalso be used and has fiber diameters ranging from 15 to 25 micrometers,and fiber lengths of about 0.85 to about 6.5 mm. The unrefinedself-bonding structural fibers typically have a Canadian StandardFreeness of +400 to +800 ml. These long self-bonding fibers mayconstitute greater than 50% of the fibrous media, by weight, preferably60-100% of the fibrous media, and most preferably 100%. Optionally, aminor portion of cellulose pulp which has been refined to a CanadianStandard Freeness of between +100 and -600 ml may be incorporated with amajor portion of the normally dimensioned cellulose pulp (+400 to +800ml). In particular, from about 1 to about 20% of the refined pulp andabout 50% to about 90% of the unrefined cellulose may be contained inthe matrix. Particulate may also be added.

When the particulate materials are millimicron-sized, it may bedesirable to use, in addition, a mixture of cationic and anionic resinsas described by assignee's U.S. Pat. No. 4,511,473, incorporated byreference herein. Alternatively, one may use a medium containing, inaddition to the millimicron-sized particles, a neutral organic polymericresin having oxygen atoms along the polymeric backbone thereof, asdescribed in the assignee's co-pending U.S. patent application Ser. No.401,361, filed on July 23, 1982, incorporated by reference herein.

Also of particular interest in the present invention is the use ofmodified cellulosic fibrous media carrying modified inorganic supportmaterials, such as for example are described in Regnier, U.S. Pat. No.3,983,299, Kirkland et al., U.S. Pat. No. 3,795,313, Kirkland et al.,U.S. Pat. No. 3,722,181, Mazarguil et al., U.S. Pat. No. 4,034,139,Talley et al., U.S. Pat. No. 4,118,316, Ho Chang et al., U.S. Pat. No.4,029,583 or Regnier, U.S. Pat. No. 4,108,603. These are allincorporated herein by reference. In particular, it is possible toderivatize siliceous particles with silanes and attach thereto variousion exchange or anchoring groups. In this embodiment then, both thecellulosic fiber and the siliceous particulate are modified, and theirinteraction provides increased anchoring and/or ion exchange capacity.The addition of particulate material tends to increase the rigidity andstrength of the fibrous media and renders it readily useful forindustrial applications, especially those involving high pressure.

PROCESS OF PREPARATION

The polymer-modified polysaccharide material of the invention can beprepared in various modes. Generally speaking, in one mode, one canfirst prepare the polymer and then condense the same through its hydroxyreacting groups (if available) to the polysaccharide. Alternatively, inanother mode, one can first react the polysaccharide with a hydroxygroup-reactive comonomer (a) followed by copolymerization with comonomer(b) and any other comonomers (e.g., crosslinking comonomers, hydrophobiccomonomers, etc.), as desired. These reactions are therefore of twotypes: (1) coupling of saccharides to hydroxy reactive groups oncomonomer (a), and (2) polymerization of polymerizable unsaturatedcompounds. The order in which these are carried out is not particularlycritical.

Still a third method of (indirectly) attaching the synthetic polyer tothe polysaccharide involves previous chemical activation of thepolysaccharide. For example, polysaccharide can be treated withoxidizing agents such as periodate, hydrogen peroxide, ceric or othermetallic oxidizing ions or the like. Reaction of the activatepolysaccharide with an amino-containing polymerizable monomeric compoundfollowed by reduction, will normally yield derivatizedpolysaccharide-carrying unsaturated functionalities along the chainthereof. These unsaturated functionalities can then serve as furtherattachment positions for conjugating the polymer thereto.

Another type of chemical activation of the polysaccharide involvesreaction with a compound such as a diepoxide or epichlorohydrin, whichyields a derivatized polysaccharide-carrying epoxy or other groups alongthe chain thereof. These epoxy or other groups then serve as conjugatingpositions on the polysaccharide chains.

The chemical activation modes of (indirect) attachment of the polymer topolysaccharide are particularly useful when introducing negative(anionic) functionalities into the polymer. This is due to the fact thatgraft polymerization, which is a common way of conferring positivecharge to polysaccharides such as cellulose, is not very effective whenattempting to confer negative charges (present in carboxy, phosphoric,sulphonic groups, etc.) thereto.

Polymerization of comonomers can be carried out by radical chain,step-reaction, ionic and coordination polymerization. Particularlyuseful is radical polymerization.

The free radical addition polymerization of radical polymerizablecomonomers is carried out with free radical initiators using the wellknown steps of initiation, addition and termination. A usual procedureis to utilize a substance or substances which produce radicals capableof reacting with the monomers. Probably the simplest of allpolymerization initiators are the organic peroxides and azo compounds.These substances decompose spontaneously into free radicals in commonorganic solvents at a finite rate, at temperatures between 50° and 140°C. For example, benzoyl peroxide decomposes into two benzoyloxy radicalsat 60° C. Another example is afforded by the azo compoundazo-bis-isobutyronitrile which similarly decomposes into radicals ateasily accessible temperatures.

The necessary energy may also be provided by irradiating the initiatorsystem with ultraviolet light. For example, initiation can be providedby irradiating the initiator system in the presence of photo initiatorssuch as benzophenone and its derivatives, benzoin alkyl ethers orderivatives, or acetophenone, with ultraviolet light. It is thennecessary that the initiator molecules absorb in the spectral regionsupplied. In this way, radicals can be generated at a finite rate atconsiderably lower temperatures than are necessary if purely thermalexcitation is used. Finally, bimolecular reactions may produce radicalscapable of initiating polymerization. Particularly important are theredox reactions, which occur in aqueous media, and involve electrontransfer processes. For example, the systems Fe(II) plus hydrogenperoxide, or Ag(I), plus S₂ O₃ ⁻⁻ are particularly important ininitiating the radical polymerization of monomers. Because of the lowtemperature of initiation, the redox initiators or photochemicallyinduced initiators are particularly preferred in the present invention.The amount of initiator is that sufficient to initiate thepolymerization reaction. Polymerization is carried out untilsubstantially all of the monomers or comonomers have been incorporatedinto the polymeric chains. This can be readily ascertained by simpleanalytical tests on the reaction mixture. Preferably, thispolymerization is accomplished almost simultaneously with or immediatelyprior to the covalent coupling of the polymer to the polysaccharides.Preferably, the coupling and polymerization are performed in the sameaqueous phase.

In one embodiment, the condensation of the comonomer (a) with thehydroxy group or groups of polysaccharide, whether carried out beforepolymerization or thereafter, is normally carried out by adjusting thetemperature of the reaction mixture, or by adding an appropriateacid/base catalyst.

The most preferred method of carrying out the process is in a "one-pot"system, using a hydroxy reactive comonomer (a). All desired comonomersand polysaccharide are added to an inert solvent system, such as, e.g.,water, alcohols, organics, and the like. The polysaccharide andcomonomers are treated under conditions which will initiatepolymerization of the comonomers. This can be accomplished, for example,by adding to a well stirred mixture a water solution of an initiatorsuch as ammonium persulfate and sodium thiosulfate, and initiatingpolymerization from about 15° C. to 40° C. Alternatively, a photolibileinitiator can be added and initiation caused by photochemical means.After stirring for a time sufficient to allow the polymerization toproceed to completion, the linking of the formed copolymer to thehydroxy groups of polysaccharide is caused by increasing the temperatureof the reaction mixture to a temperature sufficient to cause thiscondensation. In the case when the linking group on the copolymer is aglycidyl group, such temperature is normally around 80°-100° C. Reactionis then allowed to proceed at the second temperature for a timesufficient to either go to completion, or to achieve modification of thepolysaccaride to the desired capacity. The product is filtered, washedand dried for further treatment, if necessary. Unreacted monomer ispreferably washed away with alcohol, unreacted catalyst with aqueousmedia and polymer with methanol or ethanol.

Further reaction of the modified polysaccaride may be by crosslinking,activation of the ion exchange potential, as for example byquaternization of nitrogen functions, saponification of esters,ionization of acids, sulfonation, phosphorylation or oxidation ofepoxides, or other similar procedures. Quaternization, saponification,oxidation and salt formation are reactions well known to those skilledin the art, and will not be described in greater detail. Needless tosay, the reactions useful for potentiation of the ion exchange potentialof the material should not destroy the polysaccharide-copolymerlinkages. Generally, strong acid conditions should be avoided.

Quaternization of aminoalkyl functions can be carried out simultaneouslywith crosslinking by reacting the modified polysaccharide with diacylhalides or alkyl dihalides, at a ratio of 0.1 to 30 parts by weight ofthe halides per 100 parts of polysaccharide at appropriate temperature,time and solvent conditions.

Another further reaction of the modified polysaccharide materials wouldbe to anchor the affinity ligands or biologically active molecules tothe anchoring groups of comonomer (b). This reaction can be readilyaccomplished by mixing in an appropriate solvent, normally aqueous, theaffinity ligand or biomolecule to be anchored and the modifiedpolysaccharide, and carrying out anchoring for a time and underconditions sufficient to cause covalent coupling therebetween. It may benecessary to activate polysaccharide groups on comonomer (b) with suchmaterials as cyanogen halides, and to then further treat the activatedpolysaccharides with the affinity ligands or biomolecules. In thisembodiment, it is preferred to first couple the affinity ligand orbiologically active molecule to comonomer units (b), and then bind theresulting polymer or copolymer to the polysaccharide.

The reactions between the affinity ligand or biologically activemolecule and the anchoring groups on comonomer (b) are normally carriedout at temperatures of from 0° C. to 50° C., and may involve theaddition of catalysts such as acid or base, or metals, or such othermaterials as DCC. The resulting ligand- or biomolecule-containingmodified polysaccharide is washed under appropriate conditions and isready for further treatment, if necessary.

Hydrophobic comonomers (b)(iv) are normally added to a copolymerizationmixture in the presence of alcoholic solvents and/or surfactants.Washing is then carried out with alcohols.

As an illustrative example of the formation of a product under thisinvention can be described a composite of (1) cellulose, (2) a copolymerof (a) glucidyl methacrylate (GMA) and (b) diethylaminoethylmethacrylate (DEAEMA) and (3) a polyionene which has the followingrepeating units: ##STR18## wherein R⁷, R⁸, L, and M are as describedabove.

This will be used only to show the many variables which are involved inthe preparation, and which can be controlled to achieve a virtuallyunlimited number of products and resulting properties.

Step 1. Fiber dispersion and addition of monomers

Cotton linter is dispersed in water at 1% solids content--DEAEMA and GMAare added.

    ______________________________________                                        Variables                                                                     ______________________________________                                        (A)           Chemical nature and physical                                                  size of cotton;                                                 (B)           Purity of monomer;                                              (C)           Percent solid content;                                          (D)           Monomer/cotton ratio;                                           (E)           DEAEMA/GMA ratio.                                               ______________________________________                                    

Step 2. Polymer Formation

Temperature of slurry is raised to 15° C. to 40° C., followed byaddition of catalyst and reaction for 1-2 minutes.

    ______________________________________                                        Variables                                                                     ______________________________________                                        (A)           Temperature and reaction time;                                  (B)           Amount of catalyst                                              ______________________________________                                    

Step 3. Coupling of Polymers to Cotton

Temperature of slurry is raised to 80° C. to 100° C. within 25 minutes.Surfactant is added.

    ______________________________________                                        Variables                                                                     ______________________________________                                        (A)             Rate of temperature rise;                                     (B)             pH of slurry;                                                 (C)             Surfactant effect.                                            ______________________________________                                    

Step 4. Wash (1)

Four volumes of water are used to wash the product in order to removethe inorganic catalyst left in the system.

    ______________________________________                                        Variables                                                                     ______________________________________                                        (A)           Volume of water required to                                                   bring out the salt;                                             (B)           Mode of washing                                                 ______________________________________                                    

Step 5. Wash (2)

Two volumes of methanol are used to wash the product in order to removethe homopolymer and unreacted monomer.

    ______________________________________                                        Variable                                                                      ______________________________________                                        (A)          Amounts of methanol, depending                                                on reaction conditions                                           ______________________________________                                    

Step 6. Wash (3)

Four volumes of water are used to wash the product in order to removethe methanol entrapped in the fibers.

    ______________________________________                                        Variable                                                                      ______________________________________                                        (A)               Amount of water                                             ______________________________________                                    

Step 7. Acidification

The product from Step 6 is redispersed in water and 1M HCl is addedgradually to pH 4.0-4.5.

    ______________________________________                                        Variable                                                                      ______________________________________                                        (A)               Amount of water                                             ______________________________________                                    

Step 8-14: Quaternization

Step 8. Redispersal

The product from Step 6 is redispersed in water to a 1% solids content.

    ______________________________________                                        Variable                                                                      ______________________________________                                        (A)                Solids content                                             ______________________________________                                    

Step 9. Quaternization

1,6 dichlorohexane is added to the slurry in the presence of KI ascatalyst. The temperature is raised to 95° C. and refluxed for 15 hours.

    ______________________________________                                        Variables                                                                     ______________________________________                                        (A)           Quaternization agent;                                           (B)           Solvent;                                                        (C)           Reaction time and temperature                                   ______________________________________                                    

Step 10. Wash (5)

Water is used to remove the KI salt and the quaternization agent.

Step 11. Wash (6)

Methanol is used to remove excess quaternizing agent by increasing itssolubility.

    ______________________________________                                        Variable                                                                      ______________________________________                                        (A)              Degree of washing                                            ______________________________________                                    

Step 12. Wash (7)

A water wash is used to removal methanol from the system.

Step 13. Acidification

1M HCl is used to protonate remaining unquaternized DEAE.

    ______________________________________                                        Variable                                                                      ______________________________________                                        (A)         Balanced DEAE and QAE depends                                                 on the degree of quaterniza-                                                  tion carried out in the system                                    ______________________________________                                    

Step 14. Wash (8)

Excess acid is washed away.

The preferred formation of self-supporting fibrous media from themodified polysaccharide materials of the invention can be carried outimmediately after polymerization and polysaccharide modification. Inthis mode, unmasking the ion exchange groups or anchoring of affinityligands or biomolecules may be carried out on the formed sheetsthemselves. Alternatively, the fibrous media is formed after unmaskingof the ion exchange groups and/or anchoring of affinity ligands orbiomolecules. The preferred method is to form the fibrous sheets afterpolysaccharide modification, and carry out further reactions, such asunmasking and anchoring on the sheets.

The production of an effective polyionene-transformed modified substraterequires a binding method such that the chemical groups essential foranti-bacterial action are not blocked or stearically hindered, while atthe same time maximizing the presence of said groups. In the presentinvention, a preferred approach involves a two-step procedure whereinreactive groups are introduced into the polyionene molecule and thepolyionene containing reactive groups then bound to the substrate,perhaps through a linker or spacer group. In this manner, the biocidalpolyionene retains its active form after binding to the substrate, withthe tertiary structure essentially unaltered, thereby maximizing thehydrophobic and charge interaction forces for entrapment anddeactivation of the bio-organisms.

Transformation of the modified polysaccharide may proceed in a varietyof ways. Where the polyionene polymer has been synthesized to produce apolymer absent reactive groups along the polymer backbone, for example a6,10 polyionene, addition of a diamine, typically ethylene diamine, willserve to link the pendant chloride groups of the polyionene to themodified polysaccharide where the modified polysaccharide is onecontaining functional groups. A typical and preferred modifiedpolysaccharide is the cellulose-GMA of Example 3, but the hydroxy,carboxy, and amine modified substrates are also useful.

Alternatively, where the polyionene itself has been synthesized tocontain reactive groups along the polymer backbone as well as pendantreactive chloride groups, addition of a diamine such as ethylene diamineproduces superior binding to the modified substrate as a result of thegreater number of reactive sites per polymer chain.

Typically, the reaction mixture of modified polysaccharide, polyionene(2-30% based on the weight of the modified polysaccharide) and diamine(0.1-10% based on the weight of the modified polysaccharide) are reactedin aqueous solution at 25°-75° C. for a sufficient period of time topermit bonding of the polyionene to the modified substrate. Titration ofthe diamine to constancy is one means of following the course of thereaction. As is understood by those skilled in the art, the course ofthe bonding reaction will vary with conditions such as temperature,reactants, concentration, and the like.

A self-supporting fibrous matrix using the polyionene-transformedmodified polysaccharide of the invention can preferably be made byvacuum filtering an aqueous slurry of fibers and, if desired, additionalresins and modified or unmodified particulate. This forms a sheet havinguniformly high porosity, fine pore-size structure with excellent flowcharacteristics and is substantially homogeneous with respect to fiber,resins and particulate.

The vacuum filtration is performed on a foraminous surface, normally awoven wire mesh which, in practice, may vary from 50 mesh to 200 mesh,with mesh openings ranging from 280 micrometers to 70 micrometers,respectively. Finer meshes are unsuitable because of clogging problemsand/or structural inadequacy.

The sequence of adding the overall components to the slurry (modifiedfibers, other fibers, particulates, modified particulates, other resins,etc.) is relatively unimportant, provided that the slurry is subjectedto controlled hydrodynamic shear forces during the mixing process. Theslurry is normally prepared at, say, about 4% consistency and thendiluted with additional water with a proper consistency required forvacuum filtering and sheet formation. This latter consistency will varydepending upon the type of equipment used to form the sheet. Typically,the slurry is cast onto a foraminous surface, vacuum filtered and driedin the conventional manner.

The flat, dimensionally stable sheet can be of any desired thickness andis then cut to the appropriate dimensions for each type of application.Preferably, the wet sheet is dried and then cut to proper size in orderto form discs. These discs can be loaded onto an appropriately sizedcylinder column to form the desired medium. The disc and cylinder shouldpreferably be in interference fit so that the disc can be pushed intothe cylinder without distortion, but not fall under gravitational forceallowing gaps between the discs and the cylinder. After the column ispacked dry, a pump can be used to pump solvent through the elementstacked in the column. Preferably, the elements swell to form asubstantially tight edge seal to the cylinder wall. Because theindividual elements are dimensionally stable, the column is notsensitive to orientation or handling, a problem which is common withother chromatographic media, particularly of any gel type media. Atypical column is disclosed by Crowder, III et al., U.S. Pat. No.4,384,957, incorporated by reference herein.

In a preferred embodiment, the modified polysaccharide media of theinvention in fibrous form is shaped into a jelly-roll configuration, asdisclosed in copending U.S. patent application Ser. No. 505,532, filedJune 17, 1983 by Leeke et al., or in configuration similar to thatdescribed for mechanical filtration in U.S. Pat. Nos. 2,539,767 and2,539,768 to Anderson, and available from AMF, Incorporated, CunoDivision, as Micro-Klean® Filter Cartridges, and described in a brochureof the same title, 1981, herein incorporated by reference.

The jelly roll configuration is normally shaped into a cartridge.Utilization of the cartridge has several advantages. Production scaleflow rates of 200-500 ml/min can be utilized with the cartridge,depending on the application. The cartridges can be autoclavedseparately by rolling/encasing in Kraft non-shredding paper. They can behoused in special housings made of polysulphone tubing with acetyl endcaps, where the cartridge can be autoclaved. The cartridges show longterm stability with respect to their binding capacities at roomtemperature storage.

The rigidity of the matrix allows the column to be operable inunrestricted diameter for high volume processes. The column volume isvirtually unaffected by changing pH or ionic strength in the buffersolution. Such a system can be equilibrated and regenerated in a shortperiod of time, eliminating cumbersome procedures of column preparationand regeneration.

USES

The ion exchange, affinity, reverse phase, or bioactive materials of theinvention can be used in any of the well known prior art processes ofion exchange, affinity or reverse-phase chromatography, or as supportsfor bio-reactors, depending on the extent to which the polymer modifierof the polysaccharide substrate is itself bonded to by the polyionene.

The materials obtained in the present invention have unique propertiesover materials used heretofore. A binary system formed by mixingmodified polysaccharide, e.g., cellulose, with other type ofpolysaccharide, such as microcrystalline cellulose, and forming afibrous sheet (without the addition of extra particulate material) hasthe advantage of lacking silica materials, which normally showsnonspecific adsorption of proteins. A highly controllable degree ofswelling which can be readily controlled by adjusting the multiplevariables present in the system, allows the replacement of unmodifiedmicrocrystalline cellulose by other mechanical strengtheners, has lowproduction cost, and high exchange capacity or anchoring capacity, whichcan, in any event, be modified by controlling the ratio of comonomers(a) and (b).

A ternary system formed from modified polysaccharide, modified orunmodified particulate, and modified or unmodified fibers other thanpolysaccharide has the advantage of potential maximization of swelling,rigidity and capacity obtainable through varying the multiple variablespresent in the system. Flow rates can be controlled by varying the ratioof organic to particulate (especially silica) components withoutsignificant loss of capacity. In addition, such a system showsadvantages over prior art systems using nonmodified celluloses in that,in many instances, no refined pulp is necessary, since the polymerlinked on the polysaccharide will function as well as refined pulp inbridging particles to the fiber surfaces. The polymeric components inthe present system may also function as binder resins; therefore,addition of resins to the slurry can, if desired, be eliminated.

While ordinarily the prior art has relied on materials with high surfacearea to bind the maximum number of chemical groups thereon, thematerials of the present invention provide means of bindingmultifunctional groups per each polysaccharide molecule. As long asthese functional groups are made accessible for ion exchange oranchoring, the preparation is no longer limited to high surface areamaterials.

In any event, as a result of transformation of the modifiedpolysaccharide substrate with polyionene, an additional dimension ofbiocidal and bacterial entrapment results in separation matrices asabove described which serve to decontaminate biological andpharmaceutical solutions from microorganism-originated contaminants.

The polyionenes of the present invention are insolubilized by bonding tothe modified polysaccharide substrate. The polyionenes contain bothhydrophobic and charged groups for bacterial adsorption. By theinsertion of coupling groups and/or linker arms, the higher molecularweight bioadsorptive compounds are bonded to the modified substrate butstill provide a degree of flexibility thereto, and distortion of thestructural conformation of proteins in solution is avoided, therebyretaining the biological activity thereto.

In protein separations and purifications, the key factor which ought tobe avoided is possible damage to the protein molecules. In the presentinvention, this is avoided by using biocompatible materials such aspolysaccharides with only limited amounts of organic polymers. Thematerials are swellable and provide for very little denaturation ofprotein. Nonspecific adsorption of biopolymers is decreased, since bothacrylic and saccharide polymers show very low amounts thereof, and arehydrophilic in nature.

Another area of design flexibility and design control is in the possibleadjustment of the length of the acrylic polymer carrying the various ionexchange or anchoring groups. The variability of the polymer length notonly may eliminate steric hindrance due to solute or ligandaccessibility, but also minimizes leakage of the ligand from the matrix.The polymer "arm" should not be too long to avoid potential internalhydrophilic interaction, such as folding back. An "arm" of about 5 to 20atoms is generally ideal for attaching the bioligands.

By the use of well-known anchoring groups for affinity ligands orbiomolecules, the materials can incorporate all of the synthetic methodsdeveloped in prior art commercial materials, such a Sephadex® orSepharose®.

The matrix is chemically and physically stable with minimum change ofdimensional stability upon contact with salt and eluents.

Additionally, however, the presence of the polyionene provides anexceptional capacity for bacterial decontamination of the solutionsbeing processed.

The polyione-transformed modified polysaccharide separation matrices ofthis invention, also called the stationary phase below, are useful inthe same manner as the prior art separation matrices or stationaryphases.

Further, as is understood by those skilled in the art, while the abovedescription has focused on polyionene-transformed modifiedpolysaccharides as the separation matrix, any substruate may be equallysuitable for polyionene transformation so long as there exists amechanism for bonding the polyionene thereto. For example, co-pendingapplication Ser. No. 758,036, filed concurrently herewith, discloses apolyionene-transformed microporous membrane for separating contaminantsof microorganism origin from biological liquids. Essentially any and allpreviously known or yet to be discovered separation matrices whichsatisfy the physical characteristics of rigidity, porosity andstability, and which further lend themselves to polyionenetransformation are within the scope of this invention.

In one embodiment, the stationary phase, in sheet form, is used as thestationary phase in a chromatography column.

Referring to FIG. 7, the column, which may be in cartridge form,generally designated 10, is comprised of a cylindrical stationary phase12, and cylindrical tube 13, which form a cylindrical chamber 14 whichacts as a housing for the stationary phase 12. The solid stationaryphase 12 can be inserted into chamber 14 formed by a glass, metal orpolymeric tube or cylinder 13 having a diameter somewhat larger than theexternal diameter of the stationary phase 12. Suitable fluid admission,collection and monitoring systems can also be employed with the columnas in conventional analytical and preparative columns. The stationaryphase 12 is positioned within the chamber 14 and preferably has alongitudinal axis 16 coaxial with the axis of the cylindrical chamber14. Optionally, a plurality of cartridges may be placed in a singlehousing in various configurations to effect parallel and/or series flowbetween the cartridges (not shown). The solid stationary phase haschromatographic functionality and is effective for chromatographicseparation.

Referring to FIGS. 8 and 9, the stationary phase 12 is constructed of aswellable fibrous matrix 18, usually hydrophilic swellable, in sheetform which is the active media for chromatographic separation. Thechromatographic media in sheet form 18 is sandwiched between a singlenon-woven mesh 22 or plurality of mesh. The composite sheet ofchromatography media 18 and mesh 22, preferably non-woven, is spirallywound around a cylindrical core 24 having a longitudinal axis 16 to forma plurality of layers around the axis 16. The core 24 is provided with aplurality of longitudinal and axially oriented channels 21 for directingthe liquid into circumferential channels 23 which are in fluidcommunication with core 24. The mesh 22, due to the openness andthickness thereof, acts as a spacer means between each layer of media 18which permits the controlled swelling of the media and enhances thedistribution of the sample flowing through the stationary phase 12. Thecylindrical core 24 is provided with apertures 26 near the top thereoffor the flow of sample from the circumferential channels 23 into theopen interior of the core.

Referring to FIG. 10, the wound composite sheet 18 and 22 and core 24are then capped by stationary phase end caps 32 and 34. The stationaryphase end caps 32 and 34 of this subassembly are sealed by thermoplasticfusion to the core 24 and also to the ends of the composites 18 and 22.The subassembly, comprising 18, 22, 24, 32 and 34 is then slipped intochamber 14. The cylinder end cap 36 is then thermoplastically fused tothe top edge 31 of cylinder 13. The fluid or sample 42 can thus flowradially from the outside through the solid stationary phase to the openchannel 21 of core 24, since the interior and exterior are completelyseparated by the solid stationary phase and sealed off by stationaryphase end caps 32 and 34.

The preformed stationary phase end caps 32 and 34 are preferably appliedto the cylindrical solid stationary phase 12 by heating an inside faceof the thermoplastic stationary phase end cap to a temperaturesufficient to soften a sufficient amount of the stationary phase end capto form a thermoplastic seal with the ends of the core 24 and compositesheet 18 and 22. All of the edges are then embedded into the softenedmaterial. The softened material is then hardened, typically by ambientconditions, to form a thermoplastic sealing relationship between thesealing surface of the stationary phase end caps 32 and 34, the core 24and the ends of the solid stationary phase 12 to form a leak-proof seal.Such methods of applying stationary phase end caps are well known in thefiltration art. See, for example, U.S. Ser. No. 383,383 and U.S. Ser.No. 383,377, filed on May 28, 1982, to Meyering et al. and Miller,respectively. Optionally, the stationary phase end caps can be moldedintegrally in situ onto the solid stationary phase.

Stationary phase end caps of thermoplastic materials are preferredbecause of the ease of bonding, but it is also possible to usethermo-setting resins in a thermoplastic, fusible or heat-softenablestage of polymerization, until the bondings have been effected, afterwhich the curing of the resin can be completed to produce a structurewhich can no longer be separated. Such a structure is autoclavablewithout danger of destroying the fluid tight seal, the solid stationaryphase 12, and the stationary phase end caps 32 and 34. Thermoplasticresins whose softening point is sufficiently high so that they are notsoftened under sterilizing autoclaving conditions are preferred forbiomedical use. Exemplary of the plastic materials which can be used arepolyolefins.

Referring to FIG. 7, the preferred column 10 has a stationary phase endcap 34 on one end which does not open to the exterior of the subassembly18, 22, 24, 32, and 34 but is closed off. This stationary phase end cap34 can nest on the bottom end wall 44 of cylinder 13 while stillpermitting the flow of sample 42 into chamber 14 around the outside ofstationary phase 12, or this lower stationary phase end cap 34 of thesubassembly 18, 22, 24, 32 and 34 is in spaced apart relationship fromthe bottom end wall 44 of cylinder 13, thus permitting the flow ofsample 42 into the chamber 14.

The upper end of cartridge 40 has a cylinder end cap 36 which is influid communication with channels 21 of cylindrical core 24 thuspermitting the flow of fluid from the outer periphery of cylindricalcore 24 to the center of core 24 to the outside of cylinder end cap 36.The cylinder end cap 36 has molded thereon fitting 48 for fluidconnection through a collection means (not shown).

Referring to FIG. 8, prior to winding the chromatography media 18 on thecore 24, the exterior surface of core 24 may be completely wrapped witha scrim material 20. Additionally, after winding the chromatographymedia 18 on the core 24, the exterior surface thereof may be completelywrapped with mesh material 22.

FIGS. 10 through 12 depict another embodiment of the chromatographycolumn of this invention, the embodiment wherein the column is in discconfiguration, again wherein like character references indicate likeparts.

Referring to FIGS. 10-16, the column in disc configuration, generallydesignated 110, comprises an inlet housing member 112, an outlet housingmember 114, and a stationary phase 116.

The inlet housing member 112 comprises a sample inlet means 118, bafflemeans 120, and sample distribution means 122. The sample inlet means 118is in communication with the sample distribution means 122.

The sample distribution means 122 comprises plural radial distributionchannels or grooves 130 and plural concentric distribution channels 140,the radial distribution grooves 130 and concentric distribution channels140 being in communication with each other and with inlet means 118.Radial distribution grooves 130 comprise distribution groove bottomportions lying in a plane represented by line P₁ in FIG. 10 and P₁ ' inFIG. 12, and distribution groove wall portions 134a and 134b. Concentricdistribution channels 140 comprise concentric distribution channelbottom portions 142, concentric distribution channel wall portions 144aand 144b, and concentric distribution channel apex portions 146.

Optionally, the inlet housing member 112 may contain a venting means150, the function and operation of which will be defined below. Theventing means is in communication with a chamber 152. Chamber 152 isformed by inlet housing member 112 and outlet housing member 114 (seeFIGS. 10 and 16). Chamber 152 contains the stationary phase 116.

The outlet housing member 114 comprises a sample collection means 154and sample outlet means 156, sample collection means 154 being incommunication with sample outlet means 156.

Sample collection means 154 comprises plural radial collection grooves160 and plural concentric collection channels 170. Radial collectiongrooves 160 and concentric collection channels 170 are in communicationwith each other and with sample outlet means 156.

Radial collection grooves 160 comprise radial collection groove bottomportions lying in a plane represented by line P₂ in FIG. 10 and P₂ ' inFIG. 16, and radial groove wall portions 164a and 164b. Concentriccollection channels 170 comprise concentric collection channel bottomportions 172, concentric collection channel side wall portions 174a and174b and concentric collection channel apex portions 176.

Stationary phase 116 has chromatographic functionality and is effectivefor chromatographic separation. Referring to FIGS. 13, 14 and 15 inparticular, the stationary phase 116 may comprise a plurality of layersof a swellable fibrous matrix 180 in sheet form, having chromatographicfunctionality and being effective for chromatographic separation, and aspacer means 182 between each adjacent layer of swellable fibrous matrix180. This configuration is best shown in FIG. 15, a cross-sectional viewof one embodiment of the separation phase 16.

The swellable fibrous matrix 180 is preferably hydrophilic swellable andcomprises the active media for chromatographic separation. The spacermeans 182 may be typically a woven or non-woven mesh similar to mesh 22of FIGS. 8 and 9 above and is further described below. The mesh, due tothe openness and thickness thereof, acts as a spacer means between eachlayer of swellable fibrous matrix 180 and permits the controlledexpansion thereof without closing off the porous structure of the media,thereby enhancing the distribution of the sample flowing through thestationary phase 116.

As may be seen from FIG. 14, a typical manner of conforming thestationary phase 116 is to produce a "sandwich" of alternating layers ofswellable fibrous matrix in sheet form and layers of spacer means, withthe periphery of the sandwich compressed into a fluid tightconfiguration 184. Typically, the peripheral edges of alternating discsof swellable fibrous matrix 180 and spacer means 182 are joined.Preferably, the fibrous matrix 180 contains or has bonded therein athermoplastic polymeric material. Similarly, in a preferred embodiment,spacer means 182 also is made of or contains thermoplastic polymericmaterials. In this configuration, the edges may be uniformly joined byappropriate heat treating, e.g. sonic welding. As may be seen from FIG.10, in a preferred embodiment, the fluid tight peripheral configuration184 is itself contained in a fluid tight, hermetic seal formed by themating edges 186 and 188 of, respectively, the inlet housing member 112and the outlet housing member 114. In this manner, sample enteringthrough inlet means 118 must pass through stationary phase 116 prior toexiting through outlet means 156.

The disc configured chromatography column of FIGS. 10-16 is formed usingconventional and well known fabrication techniques. Typically, thestationary phase 116, a preformed "sandwich" of alternating layers ofswellable fibrous matrix and spacer means, with peripheral edgessonically welded and configured as in FIG. 16, is placed in inlethousing 112 and outlet housing member 114 is placed thereover.Subsequently, the mating edges 186 of the inlet housing member 188 andof the outlet housing member 190 are joined to form an airtight andfluid tight seal. In one embodiment, the edges are sealed by sonicallywelding same, the technique described in Branson Sonic Power Company,Danbury, Conn., Information Sheet PW-3, 1971, incorporated by referenceherein.

Vent means 150, as mentioned above, represents an optional configurationof the disc embodiment of the column. Its purpose is to allow air in thecolumn to exit the column during use. Typically, vent means 150 isadapted to be sealed off when all air has been removed from the system.In an alternative embodiment, vent means 150 contains a hydrophobicmedia which will allow the passage of gases but not liquids, asdisclosed in U.S. Pat. No. 4,113,627, incorporated herein by reference.

In a preferred embodiment, depicted by FIG. 16, chamber 152 is radiallyoutwardly expanding. By the term "radially outwardly expanding" is meantthat the volume at the interior chamber is less than the volume at theperiphery of the chamber. In this configuration, referring to FIG. 16,the distance between distribution means 112 and collection means 114 atthe interior, d₁, is less than the distance between distribution means112 and collection means 114 at the periphery d₂.

Because the stationary phase 116 is hydrophilic swellable, samplesolution on contact with separation phase 116 causes the separationphase to swell. As the separation phase swells, the pressuredifferential between the inlet and outlet sides of the separation mediaincreases, thereby restricting sample flowthrough. By designing ahousing as described above, i.e. in radially outwardly expandingconfiguration, the pressure differential between the inlet and outletsides of the stationary phase decreases towards the periphery, therebymaximizing utilization of the chromatographic separation function of thestationary phase and substantially increasing the adsorption capacity ofa given unit.

In another preferred embodiment, also depicted in FIG. 16, the volume ofeach succeeding concentric distribution channel 140 and concentriccollection channel 170 increases from the interior to the periphery ofthe chromatographic column. In this manner, clogging of the channels bythe swelling of the hydrophilic swellable stationary phase is vitiated,thereby promoting uniform distribution of sample and maximum utilizationof column capacity.

In the embodiment depicted in FIG. 16, lines A, A', C and C' are lineswhich represent cross-sectional view of parallel planes which areperpendicular to the longitudinal axis L of the chromatography column.Lines B and B', respectively, represent cross-sectional views of planeswhich are substantially tangent to the apices 146 and 176 of concentricdistribution channels 140 and concentric collection channels 170. PlanesB and B' form angles α and α' with planes A and A'. Thus, planes B andB', at angles α and α' to planes A and A', respectively, define aradially outwardly expanding chamber 152, which in turn defines thelimits of expansion of stationary phase 116. As described above, theoptimal configuration for the radially outwardly expanding embodiment issuch that stationary phase 116, in maximally swelled status, is justtouching the most peripheral apices 146 and 176. It is to be understoodthat angles α and α' may be the same or different and may vary with thenumber of layers of swellable fibrous matrix and the particular matrixin use. Typically, α and α' are about 21/2°.

Lines D and D', respectively, represent cross-sectional views of planeswhich contain concentric distribution channel bottom portions 142 andconcentric collection channel bottom portions 172 and define angles βand β' with planes C and C'. Thus, planes D and D', at angles β and β'to planes C and C', respectively, define the slope of the increasingdepth of channels 140 and 170. In the embodiment of FIG. 10, β and β'are typically each about 5°. However, these angles may be varied, bothwith respect to one another and absolutely.

In similar manner, it is within the scope of the present invention toconfigure a chromatographic column such that radial distribution grooves130 and/or radial collection grooves 160 increase in volume from theinterior to the periphery of the column. Such a configuration isdisclosed in U.S. Pat. No. 3,361,261, incorporated by reference herein.

As is understood by those skilled in the art, it is desirable tominimize the hold-up volume of a chromatographic column. With this inmind, an optimal design for a radially outwardly expanding chamber isthat where the distance d₂ is such as to allow the swellable stationaryphase to swell to its maximum, but with no unused space left. In thismanner, the pressure differential at the periphery is minimized, whileat the same time reducing hold-up volume to its lower limit as well.This housing configuration permits as well the use of a single layer offibrous matrix or a plurality of layers of fibrous matrix with no spacermeans interposed between layers. The radially outwardly expandingchamber coacts with the thus configured stationary phase to uniformlydistribute sample thereacross.

In order to provide a chromatographic media matrix which is coherent andhandleable, it is desirable that at least one of the components which gointo forming the porous matrix be a long, self-bonding structural fiber.Such fiber gives the stationary phase sufficient structural integrity inboth the wet "as formed" condition and in the final dry condition. Sucha structure permits handling of the phase, in particular a sheet, duringprocessing and at the time of its intended use. Preferably, the sheetswhich form the chromatographic media are formed by vacuum felting anaqueous slurry of fibers. The sheets may also be pressure felted orfelted from a non-aqueous slurry. The sheet shows a uniform highporosity, with excellent flow characteristics, and is substantiallyhomogeneous. In general, the media can range in thicknesses from about 5mils to about 150 mils (dry); however, thicker or even thinner media maybe utilized provided the sheet can be sprially wound or layered toproduce a column which can perform as described above. The media canswell to at least 25% this thickness, and generally greater, e.g. two tofour times, this thickness.

It is important when constructing the chromatography column of thisinvention that the chromatographic media used in the column be ofuniform thickness throughout its length and width and that the mediahave a substantially uniform density throughout. It is preferred thatthe layer of media be substantially homogenous with respect to itself;however, for certain applications and material, it is understood thatnon-homogeneous construction may be employed.

Since the solid stationary phase is intended in use to effect separationby maintaining a substantial pressure differential across the solidstationary phase, it is essential that the solid stationary phase have asufficient degree of compressive strength to withstand deformation undersuch loads as may be imposed upon it. Such compressive strength must notonly exist in the media itself but in the spacer means and the internalcore upon which the chromatography media, or solid stationary phase iscompressed.

Due to the swellability of the media, a key element of this invention isthe spacer means between each layer of the media and/or the coaction ofthe chamber wall and the fibrous matrix. The spacer means permitscontrolled expansion of the media and enhancement of the distribution ofsample flowing through the stationary phase. The spacer means locatedbetween each layer of the swellable chromatographic media provides forthe distribution movement of the sample as the sample passes through thesolid stationary phase. The spacer means functions to uniformly controlthickness and density of the chromatographic media during use. Inaddition, the spacer means can serve as a backing or support for thelayer of chromatographic media. This latter aspect is particularlyuseful during the manufacturing phase.

It is preferred that the spacer means be composed of a material which isinert with respect to the chromatographic process. By inert, it is meantthe material does not adversely affect the function of the solidstationary phase.

Referring to FIGS. 8 and 9, the spacer means comprises the mesh 22.Alternatively, where the column design is as depicted in FIGS. 10-16,the spacer means 182 may also comprise a mesh, or scrim and mesh. Ascrim material can function to channel, to a certain extent, the sampleflowing through the media and substantially evenly disperse the sampleaxially and circumferentially across the media. The mesh materialprovides spacing between the media to permit controlled expansionthereof to prevent the "cut-off" to flow therethrough by compression ofthe permeable media and also assists in distributing or channeling thesample flowing through the media.

The mesh material is an open type of material having openings ranging,for general guidance, from 1/16 inch to 1/4 inch.

It should be noted that the thickness of the spacer means, i.e. thescrim and particularly the mesh material, and the pore size of each tobe used may be readily determined by one skilled in the art byperforming tests which vary these factors. Such factors as the opennessand thickness of these spacer means are highly dependent on the type ofmedia utilized, e.g. swellability, wettability, thickness, chemicalcomposition, etc., the flow rate of the sample through the stationaryphase, the surface area of the stationary phase, e.g. number ofwindings, thickness of media, diameter of stationary phase, etc. Is isthus very difficult to clearly specify these variables, other than tosay that these may be determined by either trial and error or moreelaborate testing procedures to determine the optimum parameters.

The preferred mesh material, at this time, is polypropylene CONWED(Grade TD-620).

The overall width of the stationary phase in accordance with the presentinvention can be infinite, the actual diameter being limited only bypractical considerations such as space requirements. Since the diameteror width of the overall column can be increased without theoreticallimitation, the sample size or amount of substance to be separated inthe bed is not limited. Thus, the diameter can be increased to separatethe desired amount of sample substance to be produced.

In operation, the sample is driven through the stationary phase andseparated into distinct chromatographic fractions by the chromatographicmedia. The spacer means induces and permits flow of this stream as itmoves through the column and therefore provides for improved resolutionand utilization of the media's potential capacity.

Referring to FIG. 7, the sample is preferably introduced at the bottomof the column flowing to the outer surface of the solid stationary phaseand then flowing radially inward through the layers of chromatographicmedia and spacer means into the channels 21 of core tube 24 and iswithdrawn centrally. It is apparent, from what has been set forth above,that the radial flow can also be caused to circulate in the oppositedirection.

Referring to FIG. 10, sample is preferably introduced at the inlet 118,passes to distribution means 122, is substantially uniformly distributedover the surface of the stationary phase 116 by radial distributiongrooves 132 and concentric distribution channels 130, and passes throughradial collection grooves 140 and concentric collection channels 170 andexits through outlet 156.

The chromatographic columns of this invention may be used for any of thewell-known chromatographic separations usually performed withconventional columns. Additionally, the columns of the present inventionmay be found useful in the areas where conventional columns areimpractical.

The novel columns of this invention can be used for separations in theanalytical and preparative fields. The columns can be connected to allcommon types of chromatographic equipment. Several columns or cartridgesof solid stationary phase can be connected in series or parallel. Inlarge units, the columns can contain identical or differentchromatographic media and can be of identical or different length and/ordiameter. See for example Daly et al., application Ser. No. 611,662,filed May 18, 1984, incorporated by reference herein. It has been foundthat the aforedescribed stationary phase produces unexpected results inthat the flow of sample through the column is enhanced withoutdestroying the adsorptive capacity of the media. Additionally, whenprotein and dye staining tests were performed it was found that thestationary phase of this invention provided even distribution of sampleflow therethrough without an increase in pressure drop when compared toa stationary phase not utilizing the spacer means described herein.

The stationary phases decrease total processing time and when used withthe proper chromatographic media has excellent binding capacity. Thestationary phases may be used with standard type pumps, gravity feed, orsyringes, utilized, in their preferred mode, at from 1 to 50 PSI, andeven under vacuum. The stationary phases of chromatographic media aretotally enclosed and completely self-contained to ensure sterileconditions. Due to the fact that the solid stationary phase ismanufactured in a factory and assembled therein, each is virtuallyidentical to the other, does not vary as in previously known columns andeliminates the dependence upon packing expertise.

It has surprisingly been found that when a column configured as in FIG.16 is employed, the actual capacity is substantially increased over thatof the column of FIG. 10. In this way, the actual capacity more closelyapproximates the theoretical capacity of the column. By configuring thecolumn to maximize sample distribution, minimize hold-up volume, andmaximize stationary phase utilization by creating a differentialpressure gradient which decreases from the interior to the periphery,the useful and effective life of the column is substantially improved.

EXAMPLES

Having now generally described this invention, the same will be betterunderstood by reference to certain specific examples which are includedherein for purposes of illustration only, and are not intended to belimiting of the invention unless otherwise specified.

Example 1 Poly(diethylaminoethyl methacrylate)-g-Cellulose

(a) Recipe

    ______________________________________                                        Reagent                  Quantity                                             ______________________________________                                        Microcrystalline cellulose                                                                             10     g                                             Diethylaminoethyl methacrylate                                                                         25     cc                                            Glycidyl methacrylate    2.5    cc                                            Ammonium persulfate      1      g                                             Sodium thiosulfate       1      g                                             Water                    500    cc                                            ______________________________________                                    

(b) Procedure

1. Cellulose was well dispersed in water in a reactor.

2. Diethylaminoethyl methacrylate and glycidyl methacrylate were wellmixed before pouring into the reactor.

3. After pouring the monomers into the reactor, the mixture was stirredfor 5 minutes.

4. Ammonium persulfate and sodium thiosulfate were dissolved in 20 mlwater; and then poured into the reactor.

5. The reactants were stirred for 20 minutes at 15° C. to 40° C.; thetemperature was then increased to 80° C.

6. Stirring was maintained for 1 hour in the range of 80°-90° C.

7. A period of 0.5 hour was allowed to cool down the products.

8. The product was filtered and washed well with water and acetone.

(c) Results

The number of available DEAE functional groups was determined bytitrating with 0.1M HClO₄ in glacial acetic acid (0.1M HCl in aqueoussolution) on Brinkman Potentiograph E 536. The instrument was calibratedby measuring commercial DEAE cellulose as the control, and capacity wasexpressed as milliequivalent (mEQ) per gram of dry material. Thecopolymerized cellulose showed approximately three times more capacitythan that of the cellulose made from the conventional prior artderivative method.

The results were further confirmed by the measurement of albuminadsorption capacity. This was done by preparing a fibrous sheet, cuttinginto discs and packing in a 76 mm size column. Albumin in phosphatebuffer solution was pumped through the column and later eluted with 1NNaCl solution. The amount of albumin measured at 280 nm O.D. showed thefollowing results (Table 1):

                                      TABLE 1                                     __________________________________________________________________________    Beaker Test on DEAE Media                                                                 pH                    Capacity Test                               Sample                                                                            Sample Weight                                                                         Media                                                                              0.1 M NaOH                                                                           Media                                                                              BSA  A280                                        No. (Dry/Wet)                                                                             in Buffer                                                                          Added  In BSA                                                                             Conc.                                                                              t = 1 hr                                                                           (Mg/g)                                 __________________________________________________________________________    1   1/9.9   5.63 2.7 ml 6.25 1030 mg                                                                            0.25 991                                    2   1/8.7   5.5  3.2    6.25 1030 0.33 978                                    3   1/8.33  5.68 2.8    6.25 1025 0.38 966                                    4   1/7.69  5.68 2.8    6.25 1025 0.69 918                                    5   1/7.69  5.68 2.8    6.25 1895 4.5  1195                                   6   1/8.7   5.57 3.9    6.30 1025 0.19 995                                    7   0.88/8.0                                                                              5.5  3.6    6.30 1533 1.49 1298                                   8   1.0/8.33                                                                              5.52 3.3    6.25 1030 0.36 974                                    9   1.0/8.33                                                                              5.52 3.3    6.25 1236 0.97 1085                                   __________________________________________________________________________

Example 2 Quaternized Poly(diethylaminoethyl) methacrylate)-g-Cellulose

(a) Recipe

    ______________________________________                                        Reagent                   Quantity                                            ______________________________________                                        Poly(diethylaminoethyl methacrylate)-                                                                   5      g                                            g-cellulose (Example 1)                                                       1,6-Dichlorohexane or 1,4 3      cc                                           dichlorobutane                                                                Potassium iodide          0.1    g                                            Isopropanol               100    cc                                           Water                     100    cc                                           ______________________________________                                    

(b) Procedure

1. A round neck flask was filled with wet poly (diethylaminoethylmethacrylate)-g-cellulose, 1,4-dichlorobutane, potassium iodide andisopropanol.

2. The reaction mixture was refluxed overnight.

3. The product was filtered and washed well with acetone and water.

4. The sample was acidified with 10⁻² N HCl, then washed well withwater.

(c) Results

The results demonstrate the effectiveness of 1,6-dichlorohexane ascross-linker on fixing the charged groups. 1,6-dibromo or diiodo hexanehave also been applied as cross-linkers with success.

To improve quaternization percentage, water soluble quaternizationreagents, halo compounds, such as 1,3-dichloro-2-propanol,1-chloro-2-propanol, chloroacetic acid, methyl chloroacetate andchloroethyl diethylamine can be applied with success. The quaternized(QAE) media derived from ethyl iodide showed exceptionally high BSAbinding capacity in the pH range from 7 to 8.5. The results are shown inTables 2 and 3.

                  TABLE 2                                                         ______________________________________                                        Quarternization Percentage in QAE                                             Media Derived from Different Q-Reagents                                       Sample No.   Q-reagent       Q (%)                                            ______________________________________                                        QAE-1        1-chloro-2-propanol                                                                           13                                               QAE-2        1,2-dichloro-2-propanol                                                                       77                                               QAE-3        methyl chloroacetate                                                                          83                                               QAE-4        chloroethyldiethylamine                                                                       82                                               QAE-5        ethyl iodide    80                                               ______________________________________                                    

                                      TABLE 3                                     __________________________________________________________________________    BSA Capacity of Various QAE Media                                             vs. pH of Phosphate Buffer Solution                                           Sample          BSA       BSA       BSA                                       No. Q-reagent   Cap. (Mg/g)                                                                          pH Cap. (Mg/g)                                                                          pH Cap. (Mg/g)                                                                          pH                                 __________________________________________________________________________    QAE-1                                                                             methyl chloroacetate                                                                      1527   6.29                                                                             1027   7.36                                                                             758    8.69                               QAE-2                                                                             1-chloro-2-propanol                                                                       1376   6.30                                                                             671    7.30                                                                             336    8.12                               QAE-3                                                                             1,2-dichloro-2-propanol                                                                   1466   6.25                                                                             676    7.29                                                                             387    8.18                               QAE-4                                                                             ethyl iodide                                                                              1391   6.27                                                                             1015   7.59                                                                             816    8.26                               QAE-5                                                                             1-chloro-2,3-propanediol                                                                  1397   6.28                                                                             692    7.33                                                                             367    7.98                               QAE-6                                                                             chloroethyl diethylamine                                                                  1483   6.27                                                                             559    7.39                                                                             290    8.60                               DEAE                                                                              --          1625   6.38                                                                             738    7.32                                                                             296    7.98                               __________________________________________________________________________

Example 3 Preparation of Cellulose-GMA Matrix

(a) Formulation

    ______________________________________                                        Reagent                Quantity                                               ______________________________________                                        Refined cellulose      5.0    g                                               Glycidyl methacrylate  12.5   ml                                              APS                    0.5    g                                               STS                    0.5    g                                               D.I. H.sub.2 O         250    ml                                              ______________________________________                                    

(b) Process

The cellulose was dispersed in the D.I. H₂ O with agitation and heatedto 80° C., with agitation. The glycidyl methacrylate, APS and STS wereadded to the reactor and the reaction permitted to proceed for one hour.The reaction was terminated and the covalently bound GMA-cellulosematrix removed, washed with 5×1.8 liters of D.I. H₂ O and stored forfurther processing. (Conversion to polyionene-transformed matrix).

Example 4 Preparation of Cellulose-GA Matrix

(a) Formulation

    ______________________________________                                        Reagent               Quantity                                                ______________________________________                                        Refined cellulose     5.0    g                                                Glycidyl acrylate     12.5   ml                                               Ethoquad C/25         0.5    ml                                               APS                   0.5    g                                                STS                   0.5    g                                                1.0 M HCl             16.67  ml                                               D.I. H.sub.2 O        250    ml                                               ______________________________________                                    

(b) Process

The cellulose was dispersed in the 250 ml of D.I. H₂ O with agitation at80° C. and the glycidyl acrylate added to the reactor. Temperature andagitation were maintained, the APS, STS and HCl added, and the reactionpermitted to proceed for one hour. The covalently bonded cellulose-GApre-ligandized matrix was removed, washed with 7×2 liters of deionizedwater and stored for further further treatment. (Conversion topolyionene-transformed matrix.)

Example 5 Cellulose-GMA Matrix Modified With Methacrylic Acid

(a) Formulation

    ______________________________________                                        Reagent               Quantity                                                ______________________________________                                        Refined cellulose     5.0    g                                                methacrylic acid      12.5   ml                                               GMA                   1.25   ml                                               APS                   0.5    g                                                STS                   0.5    g                                                D.I. H.sub.2 O        250    ml                                               ______________________________________                                    

(b) Process

The grafting-polymerization technique of Example 3 above was followed.At the end of the reaction, the matrix (CM matrix) was washed five timeswith 1.8 liters of D.I. H₂ O and stored for further processing.

Example 6

A beaker test on the capacity of media prepared according to Sample 7 ofTable 1 supra was carried out on various different types of proteinmolecules. The results are shown in Table 4 below:

                                      TABLE 4                                     __________________________________________________________________________    Beaker Test on Media                                                                           pH                   Capacity Test                           Sample                                                                            Sample Nature                                                                              Media                                                                              0.1 M NaOH                                                                           Media in                                                                           Protein                                                                           A280                                    No. (Dry/Wet)    in Buffer                                                                          Added  Protein                                                                            Conc.                                                                             t1 = hr                                                                            (Mg/g)                             __________________________________________________________________________    1   Ovalbumin PI = 4.6                                                                         5.74 5.0 ml 6.3  2038                                                                              4.37 1343                                   Crude dried                                                                   egg white m.w. = 43,000                                                       50 mg/ml                                                                  2   BSA PI = 4.9 5.63 4.0    6.3  1811                                                                              3.07 1327                                   (50 mg/ml m.w. = 65,000)                                                  3   Soybean PI = 4.15                                                                          5.52 5.0    6.3   443                                                                              1.55  200                                   Trypsin m.w. = 20,100                                                         Inhibitor                                                                     (10 mg/ml)                                                                4   Amylo- PI = 3.5                                                                            5.60 4.1    6.3   954                                                                              4.25  282                                   glucosidase                                                                   (20 mg/ml)                                                                5   Pepsin PI = 2.2                                                                            5.63 3.9    6.3  1744                                                                              2.52 1073                                   (50 mg/ml)                                                                    m.w. = 34,500                                                             __________________________________________________________________________

Example 7 Plasma Fractionation Using the Media of Example 2

775 ml of Cohn fractions II and III from human plasma were dissolved in0.01M phosphate buffer at pH 6.5. This solution was added to a column(7.7 cm i.d.×4.3 cm length, vol=200 ml) containing 25 g of the media ofExample 2. 2.7 g of IgG were recovered from the non-bound fractionswhereas elution of bound material with 1M sodium chloride yielded 7.5 gof albumin.

Example 8 Formulation of a Sheet Containing Both Modified Cellulose andModified Silica

(a) Silanization of Silica

The silanization process can be performed either in toluene or in water.The reaction mechanism involves condensation of the halide or silanolfunctional groups on the organo-silane with silanols on the silicasurface. Therefore, the reaction conditions depend very much on thenature of the silane and the surface property of silica. The selectionof silica is made based on both chemical and physical factors.Chemically, it should have a surface property favorable for silanizationreactions; physically, the particle size should be large enough topermit the least amount of pressure build-up in a column up to 2 ft.length, as long as the composite structure homogeneity can be maintainedin the formulation. The following three grades of silica gel fromDavidson Chemicals are the choice to meet such requirements:

    ______________________________________                                              Approx.                                                                       Particle   Surface  Pore    Pore pH                                           Size       Area     Vol.    Dia. 5%                                     Grade (Micron)   (m.sup.2 /g)                                                                           (cc/g)  A    Slurry                                 ______________________________________                                        922   50         750      0.43    22   4.0                                    950   30         600      0.43    25   6.0                                    952   70         320      1.50    250  7.0                                    ______________________________________                                    

The maximum pore diameter from Davidson's product is around 250 A, whichcan only accommodate protein molecules smaller than albumin. Controlledpore glass of 1000 A or controlled pore silica of 500 A needs to be usedto facilitate the diffusion of larger protein molecules such as IgG orimmune complex. DEAE or SP are introduced onto silica gel through thefollowing route: ##STR19##

(b) Formulation of the Slurry

The modified cellulosic fiber from Example 2 and the silanized silicafrom (a) were mixed in a tank at 1 to 2% consistency to form a slurryaccording to the following formulations (Table 5):

                  TABLE 5                                                         ______________________________________                                              Modified  Refined         DEAE                                                (Long     Pulp            Silica                                        Sample                                                                              Cellulosic)                                                                             (+40 CSF)       952   %                                       No.   Fiber (%) %               %     Retention                               ______________________________________                                        1     20        10              70    90                                      2     30         0              70    80                                      3     30        20              50    95                                      4     50         0              50    90                                      5     20        7%(+40) & 7% (-10)                                                                            60    100                                     ______________________________________                                    

Alternatively, copolymerization can be performed on the mixture of largeand small refined pulp in the same reactor. Silica 952, being large insize (70 micron or larger), can be held by the modified cellulose alonewithout refined pulp. No binder is required, since the polymer oncellulose is also functional as a binder.

(c) Formulation of a Column

The slurry was cast onto a foraminous surface, vacuum felted, and driedin a conventional manner. The flat, dimensionally stable sheet was thencut to the appropriate dimensions for each type of column. The cut discswere stacked in the cylinder in an appropriate height.

(d) Discussion and Results

The above prepared matrix was cut to 9.0 mm diameter sized discs andstacked to 6-inch length with 0.85 grams of dry weight material. Afterfollowing the swelling, adsorption and elution procedures, the albuminadsorption capacity was measured and the number of DEAE groups wastitrated, with the results shown in the following Table 6:

                  TABLE 6                                                         ______________________________________                                        MATRIX               CAPACITY                                                 CHARACTERIZATION     By        Albumin                                        Exp           +40 Refined                                                                              Silica                                                                              Titration                                                                             Adsorption                             No.  Modified Pulp       952   (mEQ/G) (mg/g)                                 ______________________________________                                        1    20%      10%        70%   0.89    171                                         (Inact.) (Inact.)   (Act.)        182                                    2    Act.     --         Act.  2.0     245                                                                           249                                    3    Act.     --         Inact.                                                                              1.0     120                                                                           123                                    4    Inact.   Inact.     Inact.                                                                              0        0                                     5    100%     --         --    2.0     264                                         Act.                              270                                    ______________________________________                                    

The results fully demonstrate the contribution of the ion exchangefunctional groups from the organic matrix. The enhanced capacity isachieved by making cellulose and binders all contributing theiravailable sites for ion exchange, in addition to silica.

Example 9 Preparation of an Affinity Chromatography System

Component A: cellulosic fiber

Component B: glycidyl methacylate

Component C: glucose

Method A

Step 1. Coupling of C to B ##STR20## Step 2. Formation of Polymers withControlled Ratio of B to BC

    ______________________________________                                        Component B (10%) polymerization                                                                     Copolymer                                              +                      of 10% B                                               by Redox catalyst      & 90% BC                                               Component BC (90%)                                                            ______________________________________                                    

Step 3. Coupling of the Above Copolymer to Component A

With excess amount of catalyst left in Step 2, the epoxy groups inComponent B of the above copolymer can be coupled to cellulose, byraising the temperature to 90° C. The chemical reaction is exactly thesame as Step 1, except that the Component A is in polymeric form whereasComponent C is a monomer.

Method B

Step 1. Formation of Acrylic Copolymer ##STR21## Step 2.

Component C is added to the polymer on a proper molar ratio such that90% of the available epoxy groups will be reacted with C under acid orbase catalytic conditions. The remaining 10% will be left available forcoupling on to the cellulose surface afterwards.

Example 10 Formation of Anionically Modified Cellulose According to theInvention

1. Cellulose Activation

Cellulose was reacted with potassium periodate in 2-5% water dispersion,with the periodate being at 2% w/v at pH 3.0 for about 2 hours at roomtemperature (or 30 min at 40° C.) while stirring constantly. Theoxidized cellulose was vacuum felted on a screen and washed withdeionized water until removal of the unreacted oxidants was complete.The conductivity of the washing water was measured until no more saltcame off from cellulose.

2. Monomer Coupling to the Activated Cellulose

The activated cellulose was redispersed in deionized water at 2%consistency, and amino ethyl methacrylate (AEM) was added. The weightratio of AEM to cellulose was approxiately 25%. The temperature wasraised to 50° C. and AEM was allowed to react with the activatedcellulose for 1 hour under agitation.

3. Formation of Copolymer

Glycidyl methacrylate (GMA) was then added at a weight ratio of 9 to 1of AEM, while maintaining the reactor at 50° C. Nitrogen gas was bubbledinto the reaction tank to remove oxygen gas dissolved in the aqueoussystem. Catalyst, such as ammonium persulfate and sodium thiosulfatemixture or organic peroxide, was added to initiate the polymerizationreaction via the vinyl groups for 10 minutes. The reaction kineticscould be followed by measuring the solution turbidity or the decrease ofglycidyl groups in the liquid.

4. Conversion of the Glycidyl Groups to the Corresponding Anionic Groups

The conversion of epoxy groups to carboxyl groups was performed by usingpotassium permanganate as oxidant at 60° C. for 4 hours. The finalproduct was thoroughly washed with deionized water and methanol forremoving unreacted species and homopolymers.

5. Fabrication of Chromatographic Columns

The materials thus prepared were formulated by vacuum felting to formcomposite filter pads as shown in Table 7.

                                      TABLE 7                                     __________________________________________________________________________    Formulation of Anionically Modified Pads                                                                   Polymer Grafted on M.C..sup.2                                 Polymer Grafted on Fiber                                                                      Powder Cellulose                                      Natural Cellu-                                                                        Type            Type                                                  losic Fiber                                                                           Cotton          M.C. of                                                                             Wt. of Binder Resin                        Filter                                                                             Soft Cotton                                                                           Linter Wt. of polymer/                                                                        90 micron                                                                           polymer                                                                              Ethylene Glycol                     No.  Linter.sup.1                                                                          with GMA                                                                             Wt. of Fiber                                                                           size.sup.3                                                                          Wt. of M.C.                                                                          Diglycidyl Ether                    __________________________________________________________________________    Cat #1                                                                             15%     40%    2.0      40%   1.0    5%                                  Cat #2                                                                             20%     40%    1.5      35%   0.5    5%                                  Cat #3                                                                             10%     50%    1.0      35%   0.5    5%                                  __________________________________________________________________________     .sup.1 Southern Cellulose Co.                                                 .sup.2 M.C. = microcrystalline                                                .sup.3 From FMC, Grade pH 102.                                           

The balance of pad capacity and flow rate can be adjusted, based uponthe following reaction variables:

(a) size of cotton fibers and microcrystalline cellulose powder,

(b) the amount of polymers grafted onto cellulose,

(c) the structure and nature of the polymers,

(d) the nature and the amount of the oven-dried sheets can be cut intoproper sizes and can be stacked into columns for chromatographicseparation of protein molecules.

Capacity Test Results

The capacity of a column with this material was measured from the amountof bovine gamma globulin (BGG) adsorbed on the column under specificconditions, and was expressed as milligrams of BGG adsorbed per gram ofcolumn material. The procedure of the test was as follows:

(a) A peristaltic pump was connected to the column with a pressure gaugeinstalled in the line and a UV monitoring unit, to follow the proteinconcentration at 280 NM.

(b) Column Packing: A column packed with 25 mm dia. disks and 6-inch inheight will generally have pressure across the column in the range of 1to 10 psi, depending on the flow rate and pad porosity. The pads shouldbe packed snugly so there is very little gap between them but not jammedwith great force to fragment their structure. The total amount of padsinstalled in the column was weighed so the total capacity could beexpressed either on the column volume or on a weight basis.

(c) Column Equilibration: The equilibration buffer was pumped throughthe column until the effluent had the same pH and conductivity as thestarting solution. About 5 volumes of buffer were needed to reach theequilibrium conditions.

(d) Adsorption of BGG by the Cationic Exchange Column: Based on anaverage BGG adsorption capacity around 200 mg/g of pad, a volume of BGGsolution was poured into a graduated cylinder so that there were abouttwo volumes of the amount of BGG required to saturate the column. Thesolution was recycled through the column at 5 ml/min of flow. Theconcentration of BGG was monitored by the UV monitor at 280 NM. When thecolumn reached the saturation point, the A280 leveled off. The columnwas washed with equilibration buffer until A280 returned to baseline.With the extinction coefficient of BGG being=1.3, the capacity wasexpressed as follows: ##EQU1## The pH effect on the capacity of thecationic exchange filter No. Cat. #1 and Cat. #2 are shown in Table 8.

                  TABLE 8                                                         ______________________________________                                                                  Adsorption    Capacity                              Pad   Column              Condition                                                                             WP    on BSA                                Weight                                                                              Size         pH     Flow Rate                                                                             psi   Mg/g                                  ______________________________________                                        3.29 g                                                                              25 mm × 7 pads                                                                       5.5    2 ml/min                                                                              8     837                                   3.29 g                                                                              25 mm × 7 pads                                                                       6.0    2 ml/min                                                                              8     960                                   2.21 g                                                                              25 mm × 4 pads                                                                       6.5    2 ml/min                                                                              0     665                                   2.03 g                                                                              25 mm × 4 pads                                                                       7.0    2 ml/min                                                                              1     530                                   2.11 g                                                                              25 mm × 4 pads                                                                       7.5    2 ml/min                                                                              0     215                                   ______________________________________                                    

Conclusions for Example 10

A cationic exchanger with high capacity has been developed, as tested byadsorption of Bovine gamma globulin. High flow rate applicable for largescale industrial operations can be attained.

The system is basically a composite structure with cellulose fiber assupporting backbone and an acrylic polymer carrying glycidyl groupscovalently linked to the backbone. These groups are converted tocorresponding anionic functional groups through various types ofoxidation.

As long as the lignin content is kept low, the reaction betweencellulose and the polymer will proceed according to the conditionscovered in this Example.

Example 11 Cellulose Grafted with Polymethacrylic Acid

(a) Recipe

    ______________________________________                                        Reagent              Quantity                                                 ______________________________________                                        Cotton linter fiber  36        g                                              Methacrylic acid     90        ml                                             (or B-carboxyethyl acrylate)                                                  Glycidyl Methacrylate                                                                              9         ml                                             Triton X-100         54        g                                              Sodium Lauryl Sulfate                                                                              1.5       g                                              Ammonium Persulfate  9.8       g                                              Sodium Thiosulfate   9.8       g                                              Tributyl Amine       1.0       ml                                             ______________________________________                                    

(b) Procedure

1. Disperse 36 g of cotton linter fiber in 2 liters of water and addTributyl Amine.

2. Add mixed surfactants: nonionic Triton X-100 (from Rohm & Haas) andSodium Lauryl Sulfate (from Alcolac).

3. Add mixed monomers: methacrylic acid and glycidyl methacrylate, andraise temperature to 40° C.

4. Add catalyst mixture: ammonium persulfate and sodium thiosulfatepre-dissolved in water.

5. Raise temperature to 80° C. for 1 hour under strong agitation.

6. Let the reaction cool to room temperature, and wash with 4 columns ofde-ionized water.

7. Wash with methyl alcohol once, and again wash with water to removealcohol.

8. Adjust the pH by adding 0.1M alkaline solution.

(c) Results

The capacity test conducted by the beaker method by contactingpositively charged protein molecules with the media of this example givethe following results:

                  TABLE 9                                                         ______________________________________                                        Protein                                                                       Molecules    pH of Solution                                                   Adsorbed     and Buffer Nature                                                                           Capacity                                           ______________________________________                                        Bovine Gamma pH = 6.5 in   300     mg/g                                       globulin     Acetate buffer                                                   Hemoglobin   pH = 6.5 in   700                                                             Acetate buffer                                                   Lysozyme     pH = 7.4 in 0.01 M                                                                          750                                                             Phosphate buffer                                                 Bovine Serum pH = 4.6 in   85 mg/g                                            albumin      Acetate buffer                                                   ______________________________________                                    

Examples 12-16

These examples show the use of the carrier of the invention in a fibrousmatrix shaped into a jelly-roll form and in cartridge configuration.

Example 12 Separation of Protein Mixtures by DEAE Cartridges

In this example, it is demonstrated that DEAE "jelly-roll" cartridges,like columns, can be utilized to separate protein mixtures with a highdegree of resolution. Unlike columns, the cartridges have no undesirablepressure problems and can therefore be operated at a high flow rate witha low pressure drop. In fact, out of 15 cartridges tested, all unitsgave reproducible and comparable results.

Experiment A shows the separation of an artificial mixture of bovinegammaglobulin and bovine albumin. Experiment B shows the fractionationof human plasma. Cartridges (diameter 2.5 cm, height 7.5 cm) were usedin both of these experiments.

Experiment A

Protein: A mixture of two subclasses of gammaglobulin (483 mg) andbovine serum albumin (432 mg)

Buffers: Buffer A: Phosphate buffer (0.01M) at pH=6.8; Buffer B:Phosphate buffer (0.05M) at pH=6.0; Buffer C: Phosphate buffer (0.05M)at pH=6.2+1M NaCl

282 mg gammaglobulin Type I (100% pure) was eluted with 1M NaCl inBuffer A (Peak A in FIG. 3).

148 mg gammaglobulin Type II (approximately 90% purity) was eluted inBuffer B (Peak B in FIG. 3).

Albumin (95% purity) was eluted in Buffer C (Peak C in FIG. 3).

Experiment B

Protein: 10 mL plasma, pH=6.8 (adjusted pH)

Gradient elution: 0.1M phosphate buffer (pH=6.8 to 4.5)

Peak I: gammaglobulin

Peak II: transferrin

Peak III: albumin

Electrophoretic studies indicate that the fractions are at least 90%pure.

Yield and Recovery

    ______________________________________                                        Protein applied                                                               ______________________________________                                        10 mL plasma total O.D..sub.280 =                                                                       390                                                 gammaglobulin O.D..sub.280 =                                                                            59.4                                                transferrin, O.D..sub.280 =                                                                             44.0                                                albumin, O.D..sub.280 =   163.0                                               Other eluted proteins =   84.06                                                                         350.46                                              Yield =                   88%                                                 ______________________________________                                    

Example 13 Elution Bound Transferrin by pH Shift Using DEAE Cartridge

DEAE ("jelly-roll") cartridge media reduces protein binding capacitiesat a more alkaline pH than 7.0. This unique pH shift has been utilizedto elute bound proteins at higher pH without the use of salts andsubsequent dialysis or ultrafiltration.

A previous study had shown that 85.4% bound BSA was eluted with (0.1M)phosphate at pH 7.5. In the present example, a similar observation wasfound with transferrin. Transferrin was bound to a DEAE cartridge mediawith a (0.01M) phosphate buffer at pH=6.8, and eluted with (0.01M)phosphate, pH=7.5. 92% bound transferrin was eluted in one columnvolume. The remaining transferrin was eluted with (0.1M) phosphatepH=7.5 and (1M) NaCl. FIG. 4 shows the results.

Example 14 Use of DEAE and CM Cartridges for IgG Fractionation

1. DEAE Cartridge ("Jelly-Roll")

Applied Protein: Dialyzed human plasma

Binding Conditions: Phosphate Buffer (0.01M:0.9-1.2 mS) pH=6.3

Elution Conditions (continuous or step is usable):

    ______________________________________                                                [Buffer]   Conductivity                                                                             pH                                              ______________________________________                                        IgG       0.01 M       1.0 mS     6.8                                         Transferrin                                                                              0.025 M     1.75 mS    6.04                                        Albumin   0.06 M       3.85 mS    5.14                                        ______________________________________                                    

2. CM Cartridge ("Jelly-Roll")

Applied Protein: Unbound IgG from previous DEAE step

Binding Conditions: Phosphate Buffer (0.01M:0.9-1.2 mS) pH=6.0

Elution Conditions:

    ______________________________________                                               [Buffer]     pH    [Salt]                                              ______________________________________                                        IgG      0.01 M         6.3   1 M                                             ______________________________________                                    

Example 15 CM Cartridge Capacity at Different pH Values

Comparative studies were made with a CM Cartridge ("jelly-roll") andcommercial Whatman® CM-52. Details are shown on FIG. 5 and Tables 10 and11.

                  TABLE 10                                                        ______________________________________                                        Invention Cartridge                                                                                    Cartridge  Re-                                                                Bovine     covery                                    Buffer pH  Elution Condition                                                                           IgG Capacity                                                                             (%)                                       ______________________________________                                        Phosphate  Phosphate (0.05 M)                                                                          5.43    g    94                                      (0.01 M)   pH = 6.3                                                           pH = 6.6   (1 M) NaCl                                                         Phosphate  Phosphate (0.05 M)                                                                          5.96    g    96                                      (0.01 M)   pH = 6.3                                                           pH = 6.3   (1 M) NaCl                                                         Phosphate  Phosphate (0.05 M)                                                                          6.2     g    100                                     (0.01 M)   pH = 6.3                                                           pH = 6.0   (1 M) NaCl                                                         Phosphate  Phosphate (0.05 M)                                                                          3.8     g    99                                      (0.01 M)   pH = 6.3                                                           pH = 5.7   (1 M) NaCl                                                         Acetate (0.02 M)                                                                         Acetate (0.2 M)                                                                             3.9     g    100                                     pH = 5.4   pH = 6.3                                                                      (1 M) NaCl                                                         Acetate (0.02 M)                                                                         Acetate (0.2 M)                                                                             3.9     g    100                                     pH = 5.4   pH = 6.3                                                                      (1 M) NaCl                                                         Acetate (0.02 M)                                                                         Acetate (0.2 M)                                                                             3.45    g    98                                      pH = 5.0   pH = 4.0                                                                      (1 M) NaCl                                                         ______________________________________                                    

                                      TABLE 11                                    __________________________________________________________________________    Whatman cm-52 ®                                                                                    Adsorption                                                                          Elution                                                                             Bovine IgG                                                                             Recovery                        Buffer pH                                                                           Nature of Matrix                                                                       Column Dimension                                                                        Condition                                                                           Condition                                                                           Capacity (mg/g)                                                                        (%)                             __________________________________________________________________________    Phosphate                                                                           Microgranules                                                                          (a)                                                                              21 mL  2 mL/min.                                                                           Phosphate                                                                           1.71 g   42                              (0.01 M)                                                                            preswollen                                                                             (b)                                                                              16 mm (dia.) (0.05 M)                                       pH = 6.5                       (1 M) NaCl                                     Phosphate                                                                           Microgranules                                                                          (a)                                                                              21.5 mL                                                                              2 mL/min.                                                                           Phosphate                                                                           4.48 g   89                              (0.01 M)                                                                            preswollen                                                                             (b)                                                                              16 mm (dia.) (0.05 M)                                       pH = 6.0                       (1 M) NaCl                                     Phosphate                                                                           Microgranules                                                                          (a)                                                                              22 mL  2 mL/min.                                                                           Phosphate                                                                            7.8 g   87.6                            (0.01 M)                                                                            preswollen                                                                             (b)                                                                              16 mm (dia.) (0.05 M)                                       pH = 5.5                       (1 M) NaCl                                     __________________________________________________________________________

Example 16 Plasma Fractionation Using Quaternized Media Cartridges

Cartridge dimensions: 2.5 cm (dia.)×7.5 cm (height)

Protein: 10 mL plasma, pH=6.8

Gradient: Continuous phosphate buffer (0.01M), pH=7.3 to (0.2M), pH=4.5

Material

Input: 10 mL plasma, total O.D.₂₈₀ =400

Output: Total O.D.₂₈₀ =377

Yield: 94.25%

Results are shown on FIG. 6.

Example 17 Invention Media Containing Hydrophobic Groups

(a) Recipe

    ______________________________________                                        Poly(n-octylacrylate)-g-Cellulose                                             Reagent           Quantity                                                    ______________________________________                                        Refined pulp (+260)                                                                             20          g                                               n-octyl acrylate  50          ml                                              Glycidyl methacrylate                                                                           5           ml                                              Ammonium persulfate                                                                             2           g                                               Sodium thiosulfate                                                                              2           g                                               Water             933         cc                                              ______________________________________                                    

(b) Procedure

1. Refined pulp (+260) was well dispersed in water in a 3 neck, 3 literround flask.

2. n-octyl acrylate and glycidyl methacrylate were well mixed beforepouring into the reactor.

3. After pouring monomers into the reactor and mixing the reactionmixture well, ammonium persulfate and sodium thiosulfate solutions werecharged into the reactor at room temperature.

4. The reaction mixture was strongly agitated and the reactiontemperature was raised to 82° C. within 15 minutes.

5. Stirring was maintained for 1 hour in the temperature range of80°-85° C.

6. After cooling down the reaction mixture, the product was washed wellwith water.

Example 18 Invention Media Containing Chelating GroupsPoly(3-N,N-dicarboxymethyl-2-hydroxy-propyl methacrylate)-g-cellulose

(a) Recipe

    ______________________________________                                        Reagent           Quantity                                                    ______________________________________                                        Refined pulp (+260)                                                                             50          g                                               Glycidyl methacrylate                                                                           12.5        cc                                              Ammonium persulfate                                                                             0.5         g                                               Sodium thiosulfate                                                                              0.5         g                                               Sodium iminodiacetate                                                                           2           g                                               Water             250         cc                                              ______________________________________                                    

(b) Procedure

1. Refined pulp (+260) was well dispersed in 800 cc water in a 3 neckreactor.

2. After pouring glycidyl methacrylate into the reactor and mixing thereaction mixture well, ammonium persulfate and sodium thiosulfate werecharged into the reactor at room temperature.

3. The reaction mixture was strongly agitated, and the reactiontemperature was raised to 80° C. within 15 minutes.

4. Stirring was maintained for 1 hour in the temperature range of80°-85° C.

5. The reaction mixture was cooled to 60° C., and then sodiumiminodiacetate was charged into the reactor. Further reaction wascontinued for 26 hours.

6. The reaction mixture was cooled, and product was filtered and washed.

Example 19

    ______________________________________                                        Effect of Polymer Composition                                                 on Protein Adsorption Capacity                                                     Polymer                        BSA                                            Composition                                                                              % DEAEMA    Method  Adsorption                                Exp. % GMA      as Functional                                                                             of Polymer                                                                            Capacity                                  No.  as Coupler Groups      Formation                                                                             mg/g media                                ______________________________________                                        1     16%       84%         *10%      650                                                                 Surfactant                                        2    14         86          *10%      854                                                                 Surfactant                                        3    12         88          *10%    1,106                                                                 Surfactant                                        4    10         90          *10%    1,446                                                                 Surfactant                                        5     8         92          *10%    1,548                                                                 Surfactant                                        6     6         94          *10%    1,620                                                                 Surfactant                                        7     4         96          *10%      972                                                                 Surfactant                                        8     2         98          *10%      280                                                                 Surfactant                                        9    83           91.7      Without   980                                                                 Surfactant                                        10   10         90          Without 1,450                                                                 Surfactant                                        11     12.5       87.5      Without 1,650                                     ______________________________________                                         *10% surfactant (Lauryl Alcohol Ethoxylate) on the basis of cellulose         weight forms latex type polymer.                                         

The results indicate that either increasing or decreasing the GMAcomposition too much beyond the range of 4 to 12% by weight decreasesthe adsorption capacity. Values of GMA higher than about 12% cause adecrease in porosity, whereas values lower than about 4% cause losses ingrafting efficiency.

Example 20 Polyionene With OH Groups As Coupler Case A: Introducing OHthrough Amine Monomers. ##STR22##

This OH group will serve as coupler to be linked on to membranestructure. Reaction with epichloro hydrin produces an epoxy group inplace of the hydroxyl group based on the following reaction: ##STR23##

Case B: Introducing OH through Halide Monomers. ##STR24##

The polymer was precipitated out from the dimethyl formamide solventwith acetone and washed with acetone and stored in powder form.

Example 21 Polyionene With Chloro Groups As Coupler ##STR25## Example 22Polyionene With Aldehyde or Amine Groups As Coupler ##STR26## Example 23Polyionene Carrying Vinyl Groups For Grafting ##STR27##

With the introduction of vinyl groups to polyionene, the polymer may begrafted by the free radical reaction to the modified substrate.

Example 24 Incorporating Polyionenes into a Cellulosic Matrix through aCross-Linked Agent

(a) Formulation of the slurry

Cellulosic fibers were dispersed in a tank at 1 to 2% consistency toform a slurry. Polyionene of 30% concentration to the weight ofcellulose was added to the slurry, followed by addition of 1% each of 1,4 butanediol diglycidyl ether and tetraethylene pentamine ascross-linking agent. Agitation was continued for 10 minutes.

(b) Formation of TSM (thin sheet media)

The slurry was then cast onto a foraminous surface, vacuum felted, anddried in a conventional manner. The flat, dimensionally stable sheet ofpolyionene-transformed cellulose was then cut to the appropriatedimensions for each type of column.

Example 25 Covalent Bonding of Polyionene Carrying Hydroxyl Groups toGMA-Modified Cellulose

(a) GMA-modified cellulose was formed according to Example 3 tointroduce epoxy groups on cellulosic matrix.

(b) Polyionene carrying hydroxyl groups to be covalently bonded to thecellulosic matrix was formed according to Example 20 (Case A).

(c) A slurry was formed in the same manner as described in Example 24.

(d) TSM was formed in the manner described in Example 24.

Example 26 Covalent Bonding of Polyionene Carrying Chloride Groups toGMA-Modified Cellulose

(a) The GMA-modified cellulose of Example 3 was reacted with ethylenediamine at 50° C. for 1 hour.

(b) Polyionene carrying chloride groups was prepared according toExample 21.

(c) Slurry and TSM were formed in the same manner as above.

Bacterial Removal Test Example 27 Removal of Salmonella Typhimurium G-30Strain Cells in Saline Solution

(a) Each column (13 mm) contained 0.25 g media pad was washed with 30mls of saline prior to use.

(b) G-30 cells were grown in PPBE media, spun down and suspended insaline to a final OD₆₀₀ of 0.256 (1.3×10⁸ cells/ml). PPBE media was madeby mixing 10 g protease peptone #3 with 1 g beef extract and 5 g NaCl in1 liter of water.

(c) 60 mls were passed through each column at a flow rate of 1 ml/minand 10 ml fractions were collected. OD₆₀₀ readings were used todetermine the percentage of cells in each fraction.

Columns tested:

#1 Control #1--cellulose;

#2 Control #2--cellulose and diepoxide as cross-linker;

#3 Media made as example 24 with 30% polyionene;

#4 Media made as example 2 with 10% of polyionene;

#5 Media made as example 2 with 30% of polyionene;

By taking the original OD₆₀₀ reading of 0.256 corresponding to 1.3×10⁸cells/ml, the reduction of OD₆₀₀ should represent the relative amount ofbacteria cells removed by separation media. FIG. 17 shows the efficiencyof the bacteria removal by the separation media made above.

Example 28 Removal of Bacteria Salmonella Typhimurin G-30 in thePresence of Human Serum

Each column (13 mm) contained 0.25 g media and was washed with 30 mls ofsaline prior to use. Serum and bacteria: G-30 cells were diluted inserum and some C¹⁴ labeled G-30 cells were added (in case seruminterferred with OD₆₀₀ readings).

→ph=7.8

OD₂₈₀ =45.7/ml

OD₆₀₀ =0.227/ml (˜1.14×10⁸ cells/ml)

CPM/ml=15165 cpm/ml

60 mls were passed through each column at a flow rate of 1 ml/min and 10ml fractions were collected. OD₆₀₀ readings were used to determine thepercentage of cells in each fraction and OD₂₈₀ readings were used todetermine the percentage of protein in each fraction. Two separationmedia were tested, No. 1 was made according to Example 25 with 10%polyionene and No. 2 with 30% polyionene. The results shown in FIG. 18indicate that the polyionene-transformed modified cellulose workseffectively for bacteria removal in the presence of human serum. It isalso important to notice that the loss of protein components in serum isnegligibly small as shown from the OD₂₈₀ measurement, whereas theremoval of bacteria cells measured at OD₆₀₀ is quite significant underthe test conditions specified in the figure.

Example 29 Test on Bacteriocidal Effect of Polyioene Bonded Filter

The bacteria removal of the two filters tested in Example 28 werefurther tested on their ability to stop the growth of salmonellatyphimurim G-30 in the following manner.

(a) PPBE media was prepared as described in Example 27 and mixed with 15gram Agar with 0.2% galactose to form PPBE galactose plate.

(b) A sufficient number of bacteria cells were spread on the plate toproduce a lawn after an overnight incubation at 37° C.

(c) Disks of few mm size were made from the bacteriocidal filter madeaccording to Example 25, and placed on top of the galactose plates. Ifthe separation media inhibits the growth of the cells, there should be aclear ring of no growth around the disk. The more potent thebacteriocidal effect, the larger the diameter of the ring around thespot.

(d) Both the separation media showed clear ring spots, indicating thebacteriocidal effect of such media.

Example 30 Effect of Column Length on Bacterial Removal

The effect of column length on bacterial removal is shown in FIG. 19.The filter applied in this test was fabricated according to Example 25with 10% polyionene. The test conditions were conducted as specified inExample 27.

The invention now being fully described, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto without departing from the spirit or scope of the inventionas set forth herein.

What is claimed as new and desired to be secured by Letters Patent ofthe United States is:
 1. A polyionene-transformed modifiedpolysaccharide separation matrix comprising a modified polysaccharidehaving a synthetic polymer covalently coupled thereto and a polyionenebonded to said modified polysaccharide.
 2. A separation apparatus foreffecting separation of microorganism-originated contaminants from asample flowing therethrough comprising:at least one solid stationaryphase; a means for radially distributing the sample through thestationary phase; wherein the stationary phase comprises:(a) a swellablefibrous matrix in sheet form spirally wound around the longitudinal axisof the solid phase to form a plurality of layers around the axis; (b) aspacer means between each layer for permitting controlled swellingthereof and enhancing the distribution of sample flowing radiallythrough the stationary phase, and further wherein the swellable fibrousmatrix in sheet form comprises a polyionene-transformed separationmatrix of claim
 1. 3. A separation apparatus for effecting separation ofmicro-organism originated contaminants from a sample folowingtherethrough comprising:a housing; at least one solid stationary phasein said housing, comprising at least one layer of swellable fibrousmatrix; means for distributing said sample through the stationary phaseand means for collecting said sample after the sample has flowed throughthe stationary phase; wherein the swellable fibrous matrix in sheet formcomprises the polyionene-transformed modified separation matrix ofclaim
 1. 4. A separation apparatus for removing microorganism-originatedcontaminants from a sample flowing therethrough comprising:a housing; atleast one solid stationary phase in said housing, comprising:(a) aplurality of layers of sheets of swellable fibrous matrix and (b) aspacer means between each said fibrous matrix layer for controllingswelling of the matrix and enhancing the distribution of sample flowingthrough the stationary phase by substantially evenly dispersing thesample across the matrix; means for distributing the sample through thestationary phase; and means for collecting the sample after the samplehas flowed through the stationary phase, and wherein said swellablefibrous matrix comprises the polyionene-transformed modified separationmatrix of claim
 1. 5. A separation apparatus for removing contaminantsof microorganism origin from a sample flowing therethroughcomprising:(1) a housing, said housing comprising:(a) an inlet housingmember, and (b) an outlet housing member, said inlet housing member andsaid outlet housing member defining a radially, outwardly expandingstationary phase chamber; and (2) a stationary phase within saidradially outwardly expanding stationary phase chamber, said stationaryphase chamber comprising at least one layer of a swellable fibrousmatrix in sheet form, said swellable fibrous matrix in sheet formcomprising the polyionene-transformed modified separation matrix ofclaim
 1. 6. A separation apparatus for removing contaminants ofmicroorganismm origin from a sample flowing therethrough comprising:(1)a housing, said housing comprising:(a) an inlet housing member, and (b)an outlet housing member, said inlet housing member and said outlethousing member defining a stationary phase chamber; and (2) a stationaryphase within said stationary phase chamber, said stationary phasecomprising:(a) a plurality of layers of a swellable fibrous matrix insheet form; (b) a spacer means between each layer of said swellablefibrous matrix for permitting controlled swelling thereof and enhancingthe distribution of sample flowing through said stationary phase,whereinsaid swellable fibrous matrix in sheet form comprises thepolyionene-transformed modified separation matrix of claim
 1. 7. Aseparation apparatus for removing contaminants of microorganism originfrom a sample flowing therethrough comprising:(1) a housing, saidhousing comprising:(a) an inlet housing member, and (b) an outlethousing member, said inlet housing member and said outlet housing memberdefining a radially outwardly expanding stationary phase chamber; and(2) a stationary phase within said radially, outwardly expandingchamber, said stationary phase comprising:() a plurality of layers of aswellable fibrous matrix in sheet form, and (b) a spacer means betweeneach layer of said swellable fibrous matrix for permitting controlledswelling thereof and enhancing the distribution of sample flowingthrough the stationary phase,wherein said swellable fibrous matrix insheet form comprises the polyionene-transformed modified separationmatrix of claim
 1. 8. The separation apparatus of claim 5 whereinsaidinlet housing member comprises a sample inlet means and a sampledistribution means, said sample inlet means in communication with saidsample distribution means, and said outlet means comprises a samplecollection means and a sample outlet means, said sample collection meansin communication with said sample outlet means.
 9. The separationapparatus of claim 6 whereinsaid inlet housing member comprises a sampleinlet means and a sample distribution means, said sample inlet means incommunication with said sample distribution means, and said outlethousing means comprises a sample collection means and a sample outletmeans, said sample collection means in communication with said sampleoutlet means.
 10. The separation apparatus of claim 6 whereinsaid inlethousing member comprises a sample inlet means and a sample distributionmeans, said sample inlet means in communication with said sampledistribution means, and said outlet housing means comprises a samplecollection means and a sample outlet means, said sample collection meansin communication with said sample outlet means.
 11. The separationapparatus of claim 8 wherein said sample distribution means comprisesradial distribution grooves and concentric distribution channels, saidgrooves and channels being in communication with each other, and saidsample collection means comprises radial collection grooves andconcentric collection channels, said radial collection grooves andconcentric collection channels in communication with each other.
 12. Theseparation apparatus of claim 9 wherein said sample distribution meanscomprises radial distribution grooves and concentric distributionchannels, said grooves and channels being in communication with eachother, and said sample collection means comprises radial collectiongrooves and concentric collection channels, said radial collectiongrooves and concentric collection channels in communication with eachother.
 13. The separation apparatus of claim 10 wherein said sampledistribution means comprises radial distribution grooves and concentricdistribution channels, said grooves and channels intercommunicating, andsaid sample collection means comprises radial collection grooves andconcentric collection channels, said radial collection grooves andconcentric collection channels intercommunicating.
 14. The separationapparatus of claims 11, 12 or 13 wherein the volume of said concentricdistribution channels and/or said concentric collection channelsincreases from the interior to the periphery of said column.
 15. Theseparation apparatus of claims 11, 12 or 13 wherein the volume of saidradial distribution grooves and/or said radial collection groovesincreases from the interior to the periphery of said column.
 16. Theseparation apparatus of claims 8, 9 or 10 wherein said spacer meanscomprises:(a) a scrim layer for channeling the sample flow through thematrix and substantially evenly dispersing the sample; or (b) a meshlayer to provide a spacing between the layers to permit controlledexpansion thereof and assist in distributing the sample; or (c) (a) incombination with (b).
 17. The separation matrix of claims 2, 3, 4, 5, 6or 7 wherein separation matrix is polyionene-transformed cellulose. 18.The separation matrix of claims 2, 3, 4, 5, 6 or 7 wherein saidseparation matrix comprises(1) polysaccharide covalently coupled to asynthetic polymer; (2) said synthetic polymer made from at least onof(a) a polymerizable compound which has a chemical group capable ofdirect or indirect covalent coupling to said polysaccharide; and (b) oneor more polymerizable compounds containing(i) an ionizable chemicalgroup, (ii) a chemical group capable of transformation to an ionizablechemical group, (iii) a chemical group capable of causing the covalentcoupling of said compound (2) to an affinity ligand or biologicallyactive molecule, or (iv) a hydrophobic chemical group;wherein saidpolyionene comprises a water soluble polymer having polyquaternaryammonium groups separated by hydrophobic groups, said hydrophobic groupscomprising aromatic groups or alkyl groups, containing at least sixcarbon atoms.
 19. The separation apparatus of claims 2, 3, 4, 5, 6 or 7wherein said polyionene has the following repeating units: ##STR28##wherein R⁷ and R⁸ are C₁ -C₄ alkyl; L is --(CH₂)_(n) --P--(CH₂)_(m) --;M is --(CH₂)_(o) --Q--(CH₂)_(p) --, with P and Q being the same ordifferent and representing at least one of CH₂, CHA, C₆ H₄, C₆ H₃ A, C₆H₄ --CHA--C₆ H₄, or R⁹ C₆ H₂ A; wherein A is a reactive group comprisinghydrozy, epoxy, amino, halo, aldelyde or carboxy; wherein R⁹ is C₁ -C₄alkyl; and m, n, o, and p represent integers of 1 to
 20. 20. Theseparation apparatus of claims 2, 3, 4, 5, 6 or 7 wherein saidseparation matrix comprises a modified cellulose, said modifiedcellulose comprising cellulose covalently coupled to a syntheticpolymer, said synthetic polymer comprising a homopolymer of glycidylmethacrylate, said modified cellulose transformed by the bonding of apolyionene thereto, said polyionene having the following repeatingunits: ##STR29## wherein L is --(CH₂)₆ -- and M contains at least one of--(CH₂)₁₀ -- and --CH₂ --CHOH--CH₂ --.
 21. A polyionene-transformedmodified polysaccharide separation matrix which comprises:(1)polysaccharide covalently coupled to a synthetic polymer; (2) saidsynthetic polymer made from at least one of(a) a polymerizable compoundwhich has a chemical group capable of direct or indirect covalentcoupling to said polysaccharide; and (b) one or more polymerizablecompounds containing(i) an ionizable chemical group, (ii) a chemicalgroup capable of transformation to an ionizable chemical group, (iii) achemical group capable of causing the covalent coupling of saidsynthetic polymer (2) to an affinity ligand or biologically activemolecule, or (iv) a hydrophobic chemical group;said modifiedpolysaccharide having bonded thereto, a polyionene.
 22. Thepolyionene-transformed modified polysaccharide separation matrix ofclaim 21 wherein said polyionene comprises a water-soluble polymerhaving polyquaternary ammonium groups separated by hydrophobic groups,said hydrophobic groups comprising aromatic groups or alkyl groups, saidalkyl groups containing at least six carbon atoms.
 23. Thepolyionene-transformed modified polysaccharide seperation matrix ofclaim 22 wherein said polyionene has the following repeating units:##STR30## wherein R⁷ and R⁸ are C₁ -C₄ alkyl; L is --(CH₂)_(n)--P--(CH₂)_(m) --; M is --(CH₂)_(o) --(CH₂)_(p) --, with P and Q beingthe same or different and representing at least one of CH₂, CHA, C₆ H₄,C₆ H₃ A, C₆ H₄ --CHA--C₆ H₄, or R⁹ C₆ H₂ A; wherein A is a reactivegroup comprising hydroxy, epoxy, amino, halo, aldelyde or carboxy;wherein R⁹ is C₁ -C₄ alkyl; and m, n, o, and p represent integers of 1to
 20. 24. The separation matrix of claim 22 wherein said polymerizablecompound (a) has a chemical group capable of reacting with a hydroxygroup of said polysaccharide.
 25. The separation of matrix of claim 22wherein said polysaccharide is fibrous.
 26. The separation matrix ofclaim 22 wherein said polysaccharide is cellulose.
 27. The separationmatrix of claim 24 wherein said chemical group of said polymerizablecompound (a) is a hydroxy reactive O-alkylating chemical agent capableof reacting with the hydroxy group of sad polysaccharide.
 28. Theseparation matrix of claim 27 wherein compound (a) carrying saidO-alkylating chemical agent is selected from the group consisting ofacrylic anhydride, methacrylic anhydride, -iodo C₂ -C₆ alkyl ester ofacrylic acid, -iodo C₂ -C₆ alkyl ester of methacrylic acid, allylchloride, chloromethyl styrene, chloroacetoxy ethyl methacrylate,glycidyl acrylate, glycidyl methacrylate, 4,5 epoxypentyl-acrylate,4-(2,3-epoxypropyl)-methacrylate, 9,10-epoxyate arylacrylate, allylglycidyl ether and ethylene glycol-monoglycidyl ether-acrylate.
 29. Theseparation matrix of claim 21 wherein said synthetic polymer is ahomopolymer of glycidyl methacrylate.
 30. The separation matrix of claim22 wherein the polymerizable compound (b) has the formula: ##STR31##wherein R₁ is H or CH₃ ;A is CO, or SO₂ ; X is OH, OM where M is a metalion, OR² where R² is a straight or branched chain C₁ -C₁₈ alkyl group,OR³ OH where R³ is a straight or branched chain C₂ -C₆, alkyl oraromatic group, NR⁴ R⁵ or N+R⁴ R⁵ R⁶ where R⁴, R⁵ and R⁶ are the same ordifferent and are hydrogen, R² or R³ OH; or AX taken together has theformula: ##STR32## wherein Y is --CO², --CH₂ CO₂, --SO₃, --CH₂ SO₃, PO₄H, --CH₂ N(CH₂ COO⁻)₂, --CH₂ --NR⁴ R⁵, or --CH₂ N⁺ R⁴ R⁵ R⁶, or thecorresponding free acid, R² or R³ OH ester, or partial ester groupsthereof.
 31. The separation matrix of claim 22 wherein saidpolymerizable compound (b) contains a chemical group capable of causingthe covalent coupling of said polymerizable compound (b) to an affinityligand or a biologically active molecule.
 32. The separation matrix ofclaim 31 wherein said polymerizable compound (b) carries anelectrophilic functional group capable of covalent reaction withnucleophilic group of said affinity ligand or biologically activemolecule.
 33. The separation matrix of claim 32 wherein saidelectrophilic group has the formula (II): ##STR33## wherein R' and R"are the same or different C₁ -C₁₈ alkyl or alkanoyl radicals or R' andR" together form a 5-7 membered heterocyclic ring with the N atom, andR'" is a direct bond on a C₂ -C₃ alkyl radical.
 34. The separationmatrix of claim 27 wherein said chemical group capable of causing saidcovalent coupling carries an epoxy group.
 35. The separation matrix ofclaim 22 wherein the amount of said polymerizable compound (a) in saidsynthetic polymer is sufficient to cause substantial covalent couplingof the polymer to said polysaccharide, yet insufficient to causesubstantial loss of porosity of the modified polysaccharide.
 36. Theseparation matrix of claim 22 wherein said polymer contains more of saidpolymerizable compound (b) than of said polymerizable compound (a). 37.The separation matrix of claim 36, wherein the ratio of polymerizablecompound (b) to said polymerizable compound (a) is about 88-96% byweight of (b) to 4-12% by weight of (a).
 38. The separation matrix ofclaim 23 wherein L is --(CH₂)₆ -- and M is --(CH₂)₁₀ --.
 39. Theseparation matrix of claim 23 wherein L is --(CH₂)₆ -- and M is--(CH₂)₁₀ -- and --CH₂ --CHOH--CH₂ --.
 40. A process for preparing thepolyionene-transformed modified polysaccharide separation matrix ofclaim 21 comprising(1) reacting said polysaccharide with the chemicalgroup of compound (a) in said synthetic polymer under temperatureconditions sufficient to cause said covalent bonding; and (2) bonding apolyionene to the reaction product of (1).
 41. A self-supportingcellulosic fibrous matrix comprising(1) cellulose covalently coupled toa synthetic polymer; and (2) a polyionene reactively bonded to saidcellulose.
 42. The self-supporting cellulosic fibrous matrix of claim 41wherein said synthetic polymer comprises at least one of(a) apolymerizable compound which has a chemical group capable of direct orindirect covalent coupling to said cellulose; and (b) one or morepolymerizable compounds containing(i) an ionizable chemical group; (ii)a chemical group capable of transformation to an ionizable chemicalgroup; (iii) a chemical group capable of causing the covalent couplingof said synthetic polymer to an affinity ligand or biologically activemolecule, or (iv) a hydrophobic chemical group;and said polyionenecomprises a water soluble polymer having polyquaternary ammonium groupsseparated by hydrophobic groups, said hydrophobic groups comprisingaromatic groups or alkyl groups, said alkyl groups containing at leastsix carbon atoms.
 43. The self-supporting matrix of claim 42 whereinsaid synthetic polymer is homopolymer of glycidyl methacrylate.
 44. Theself-supporting matrix of claim 42 wherein said polyionene has thefollowing repeating units ##STR34## wherein R⁷ and R⁸ are C₁ -C₄ alkyl;L is --(CH₂)_(n) --P--(CH₂)_(m) --; M is --(CH₂)_(o) --Q--(CH₂)_(p) --,with P and Q being the same or different and representing at least oneof CH₂, CHA, C₆ H₄, C₆ H₃ A, C₆ H₄ --CHA--C₆ H₄, or R⁹ C₆ H₂ A; whereinA is a reactive group comprising hydroxy, epoxy, amino, halo, aldelydeor carboxy; wherein R⁹ is C₁ -C₄ alkyl; and m, n, o, and p representintegers of 1 to
 20. 45. The self-supporting matrix of claim 44 whereinL is --(CH₂)₆ -- and M is --(CH₂)₁₀ --.
 46. The self-supporting matrixof claim 44 wherein L is --(CH₂)₆ -- and M is --(CH₂)₁₀ -- and--CH_(2--CHOH--CH) ₂ --.
 47. The matrix of claim 41 which also compriseshighly refined cellulose pulp with a Canadian Standard Freeness ofbetween +100 to -600 ml.
 48. The matrix of claim 41 which also comprisesin addition a particulate substance having chromatographic or molecularseparation functionality.
 49. The matrix of claim 41 which is in theform of a sheet.
 50. A method for removing and inactivating contaminantsof a microorganism origin from a biological liquid comprising passingsaid liquid through a polyionene-transformed modified polysaccharidematrix wherein said modified polysaccharide matrix comprises(1)polysaccharide covalently coupled to a synthetic polymer; (2) saidsynthetic polymer made from at least one of(a) a polymerizable compoundwhich has a chemical group capable of direct or indirect covalentcoupling to said polysaccharide; and one or more polymerizable compoundscontaining(i) an ionizable chemical group, (ii) a chemical group capableof transformation to an ionizable chemical group, (iii) a chemical groupcapable of causing the covalent coupling of said compound (2) to anaffinity ligand or biologically active molecule, or (iv) a hydrophobicchemical group;said modified polysaccharide having bonded thereto apolyionene, said polyionene comprising a water-soluble polymer havingpolyquaternary ammonium groups separated by hydrophobic groups, saidhydrophobic groups comprising aromatic groups or alkyl groups, saidalkyl groups containing at least six carbon atoms.
 51. The method ofclaim 50 wherein said synthetic polymer is homopolymer of glycidylmethacrylate.
 52. The method of claim 50 wherein said polyionene has thefollowing repeating units ##STR35## wherein R⁷ and R⁸ are C₁ -C₄ alkyl;L is --(CH₂)_(n) --P--(CH₂)_(m) --; M is --(CH₂)_(o) --Q--(CH₂)_(p) --,with P and Q being the same or different and representing at least oneof CH₂, CHA, C₆ H₄, C₆ H₃ A, C₆ H₄ --CHA--C₆ H₄, or R⁹ C₆ H₂ A; whereinA is a reactive group comprising hydroxy, epoxy, amino, halo, aldelydeor carboxy; wherein R⁹ is C₁ -C₄ alkyl; and m, n, o, and p representintegers of 1 to
 20. 53. The method of claim 50 wherein said matrix isin the form of a sheet.
 54. The method of claim 53 wherein said sheet isin the form of a disc.
 55. The method of claim 50 wherein saidbiological liquid is peripheral blood.